Plasmids and utilization thereof

ABSTRACT

A shuttle vector is constructed by preparing a DNA region replicable in bacteria belonging to the genus  Rhodococcus  from a  Rhodococcus -derived plasmid having the nucleotide sequence set forth as SEQ ID NO: 73 and a plasmid or its DNA fragment having the nucleotide sequence set forth as SEQ ID NO: 74, and a DNA region replicable in  E. coli  from an  E. coli -derived plasmid or its DNA fragment. An aminoketone asymmetric reductase gene is inserted into the shuttle vector, transformants containing the vector are created, and the aminoketone asymmetric reductase and optically active aminoalcohols are produced.

TECHNICAL FIELD

The present invention relates to novel plasmids derived from any ofmicroorganisms belonging to the genus Rhodococcus (hereinafter referredto as “the genus Rhodococcus”) and to utilization thereof. Morespecifically, the invention relates to plasmids or their partial DNAfragments (hereinafter also referred to simply as “DNA fragments”), andto shuttle vectors, vectors, transformants, aminoketone asymmetricreductase production methods and optically active aminoalcoholproduction methods which utilize them.

BACKGROUND ART

The genus Rhodococcus is known to produce enzymes involved in nitrilemetabolism and to produce enzymes which asymmetrically reduceaminoketones. In particular, Rhodococcus erythropolis is known to havevery high aminoketone asymmetric reduction activity. Such microorganismsand enzymes act on α-aminoketones to high selectively produce opticallyactive β-aminoalcohols at high yields (for example, Patent documents 1and 5). Thus, it has long been desired to develop a host-vector systemintended for mass production of useful enzymes and the like in the genusRhodococcus. However, the development of vectors suitable for the genusRhodococcus as hosts has lagged behind. Only a few strains of the genusRhodococcus have been found with plasmids, namely Rhodococcus sp. H13-A(Non-patent document 1), Rhodococcus rhodochrous ATCC4276 (Patentdocument 2), Rhodococcus rhodochrous ATCC4001 (Patent document 3) andRhodococcus erythropolis IF012320 (Patent document 4).

[Patent document 1] WO01/73100

[Patent document 2] Japanese Unexamined Patent Publication HEI No.4-148685

[Patent document 3] Japanese Unexamined Patent Publication HEI No.4-330287

[Patent document 4] Japanese Unexamined Patent Publication HEI No.9-28379

[Patent document 5] WO02/070714

[Non-patent document 1] J. Bacteriol., 170, 638, 1988

DISCLOSURE OF THE INVENTION

Problems to be Solved by the Invention

As mentioned above, it has been desired to develop new vectors forbreeding and improve to industrially useful strains (mutant strains)from the genus Rhodococcus. In particular, self-cloning systems arepreferred from the standpoint of safety of the recombinant DNA microbesand their products which may be used as foods and additives. It is anobject of the present invention to provide novel plasmids that can beused as vectors for such a host-vector system.

It is desirable to create recombinant microbes suitable for industrialapplication from among Rhodococcus erythropolis which has aminoketoneasymmetric reduction activity. In particular, it is a first object ofthe invention to provide novel plasmids or their partial DNA fragmentswhich can be used to create such recombinant microbes.

If a plasmid such as described above can be obtained, it would becomeeasy to construct a shuttle vector that is replicable even in othermicrobes. It is therefore a second object of the invention to providenucleotide sequence data relating to DNA replication (replicationregion, etc.) necessary for construction of such a shuttle vector.

It is a third object of the invention to provide shuttle vectors thatare replicable in both the genus Rhodococcus and E. coli.

It is a fourth object of the invention to apply the shuttle vectors toan aminoketone asymmetric reductase.

Means for Solving the Problems

The present inventors carefully screened plasmids for vectorconstruction from among Rhodococcus strains, and as a result discoveredseveral novel plasmids usable as vectors for host-vector systems.

Furthermore, the present inventors found that it is possible toconstruct shuttle vectors by transferring into the aforementionedplasmids a drug resistance gene and a gene region that is replicable inE. coli. As a result there were obtained nucleotide sequence data,plasmids and shuttle vectors that achieve the objects stated above, andthe present invention has thereupon been completed.

Specifically, the present invention provides a DNA fragment, a DNA, aplasmid, a shuttle vector, a vector, a transformant, a method forproduction of an aminoketone asymmetric reductase, and a method forproduction of an optically active aminoalcohol, according to following(1) to (39).

-   (1) A DNA fragment having at least one nucleotide sequence selected    from the group consisting of the nucleotide sequences set forth as    SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.-   (2) A plasmid or a partial DNA fragment thereof, characterized by    comprising a DNA replication region having at least one nucleotide    sequence selected from the group consisting of the nucleotide    sequences set forth as SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO:    37.-   (3) A DNA fragment having at least one nucleotide sequence selected    from the group consisting of the nucleotide sequences set forth as    SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID    NO: 22.-   (4) A plasmid or a partial DNA fragment thereof, characterized by    comprising a coding region for a DNA replication-related protein    having at least one nucleotide sequence selected from the group    consisting of the nucleotide sequences set forth as SEQ ID NO: 1,    SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 22.-   (5) A plasmid or a partial DNA fragment thereof, characterized by    comprising a coding region for a DNA replication-related protein    having at least one nucleotide sequence selected from the group    consisting of the nucleotide sequences set forth as SEQ ID NO: 1,    SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 22 and    comprising a DNA replication region having at least one nucleotide    sequence selected from the group consisting of the nucleotide    sequences set forth as SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO:    37.-   (6) A DNA fragment having the nucleotide sequence set forth as SEQ    ID NO: 76.-   (7) A plasmid or a partial DNA fragment thereof, characterized by    comprising a promoter region having the nucleotide sequence set    forth as SEQ ID NO: 76.-   (8) A plasmid or a partial DNA fragment thereof, characterized by    comprising a coding region for a DNA replication-related protein    having at least one nucleotide sequence selected from the group    consisting of the nucleotide sequences set forth as SEQ ID NO: 1,    SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 22,    comprising a DNA replication region having at least one nucleotide    sequence selected from the group consisting of the nucleotide    sequences set forth as SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO:    37, and comprising a promoter region having the nucleotide sequence    set forth as SEQ ID NO: 76.-   (9) A circular plasmid characterized by comprising a plasmid or a    partial DNA fragment according to any one of (1) to (8), wherein the    numbers of restriction endonuclease cleavage sites are BamH I: 2,    EcoR I: 2, Kpn I: 1, Pvu II: 1 Sac I: 1 and Sma I: 1, and the size    is approximately 5.4 kbp.-   (10) A plasmid having the nucleotide sequence set forth as SEQ ID    NO: 73.-   (11) A plasmid or a DNA fragment according to any one of (1) to    (10), characterized by being derived from a bacterium belonging to    the. genus Rhodococcus.-   (12) A DNA fragment having at least one nucleotide sequence selected    from the group consisting of the nucleotide sequences set forth as    SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.-   (13) A plasmid or a partial DNA fragment thereof, characterized by    comprising a DNA replication region having at least one nucleotide    sequence selected from the group consisting of the nucleotide    sequences set forth as SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO:    72.-   (14) A DNA fragment having at least one nucleotide sequence selected    from the group consisting of the nucleotide sequences set forth as    SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID    NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 62    and SEQ ID NO: 69.-   (15) A plasmid or a partial DNA fragment thereof, characterized by    comprising a coding region for a DNA replication-related protein    having at least one nucleotide sequence selected from the group    consisting of the nucleotide sequences set forth as SEQ ID NO: 40,    SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID    NO: 55, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO:    69.-   (16) A plasmid or a partial DNA fragment thereof, characterized by    comprising a coding region for a DNA replication-related protein    having at least one nucleotide sequence selected from the group    consisting of the nucleotide sequences set forth as SEQ ID NO: 40,    SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID    NO: 55, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO:    69 and comprising a DNA replication region having at least one    nucleotide sequence selected from the group consisting of the    nucleotide sequences set forth as SEQ ID NO: 70, SEQ ID NO: 71 and    SEQ ID NO: 72.-   (17) A plasmid or a partial DNA fragment thereof, characterized by    comprising a coding region for a DNA replication-related protein    having at least one nucleotide sequence selected from the group    consisting of the nucleotide sequences set forth as SEQ ID NO: 40,    SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID    NO: 55, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO:    69, comprising a DNA replication region having at least one    nucleotide sequence selected from the group consisting of the    nucleotide sequences set forth as SEQ ID NO: 70, SEQ ID NO: 71 and    SEQ ID NO: 72, and comprising a promoter region having the    nucleotide sequence set forth as SEQ ID NO: 76.-   (18) A DNA fragment having at least one nucleotide sequence selected    from the group consisting of the nucleotide sequences set forth as    SEQ ID NO: 67 and SEQ ID NO: 47.-   (19) A plasmid or a partial DNA fragment thereof, characterized by    comprising a mobilization protein region having at least one    nucleotide sequence selected from the group consisting of the    nucleotide sequences set forth as SEQ ID NO: 67 and SEQ ID NO: 47.-   (20) A DNA fragment having the nucleotide sequence set forth as SEQ    ID NO: 75.-   (21) A plasmid or a partial DNA fragment thereof, characterized by    comprising a mobilization-related region having the nucleotide    sequence set forth as SEQ ID NO: 75.-   (22) A circular plasmid characterized by comprising a plasmid or DNA    fragment according to any one of (12) to (21), wherein the numbers    of restriction endonuclease cleavage sites are BamH I: 2, Pvu II: 4,    Sac I: 3 and Sma I: 4, and the size is approximately 5.8 kbp.-   (23) A plasmid having the nucleotide sequence set forth as SEQ ID    NO: 74.-   (24) A plasmid or a DNA fragment according to any one of (12) to    (23), characterized by being derived from a bacterium belonging to    the genus Rhodococcus.-   (25) A DNA fragment having the nucleotide sequence set forth as SEQ    ID NO: 77.-   (26) A DNA fragment characterized by comprising a promoter region    having the nucleotide sequence set forth as SEQ ID NO: 77.-   (27) A shuttle vector replicable in bacteria belonging to the genus    Rhodococcus and E. coli, and comprising a plasmid or partial DNA    fragment thereof according to any one of (1) to (26) and a DNA    region replicable in E. coli.-   (28) A vector characterized by being constructed using a shuttle    vector according to (27).-   (29) A vector characterized by comprising a plasmid or DNA fragment    according to any one of (6), (7), (25) or (26).-   (30) A vector according to (28) or (29), characterized by having    inserted therein an aminoketone asymmetric reductase gene.-   (31) A vector according to (30), characterized in that the    aminoketone asymmetric reductase gene is a nucleic acid coding for a    protein consisting the amino acid sequence set forth as SEQ ID NO:    78, or a nucleic acid that codes for a protein having the amino acid    sequence set forth as SEQ ID NO: 78 with a deletion, insertion,    substitution or addition of one or a plurality of amino acids, and    having aminoketone asymmetric reduction activity.-   (32) A vector according to (30), characterized in that the    aminoketone asymmetric reductase gene is a nucleic acid consisting    the nucleotide sequence set forth as SEQ ID NO: 79, or a nucleic    acid that hybridizes with nucleic acid having a nucleotide sequence    complementary to the nucleotide set forth as SEQ ID NO: 79 under    stringent conditions, and that codes for a protein having    aminoketone asymmetric reduction activity.-   (33) A transformant containing a vector according to (28) or (29).-   (34) A transformant containing a vector according to any one of (30)    to (32).-   (35) A method for production of an aminoketone asymmetric reductase,    which comprises a culturing step in which a transformant according    to (34) is cultured in medium that allows growth of said    transformant, and

a purification step in which the aminoketone asymmetric reductase ispurified from said transformant obtained in said culturing step.

-   (36) A method for production of an optically active aminoalcohol,    wherein an aminoketone asymmetric reductase obtained by the    production method of (35) is reacted with an enantiomeric mixture of    an α-aminoketone compound represented by the following general    formula (1):

wherein X may be the same or different and represents at least onespecies selected from the group consisting of halogen, lower alkyl,hydroxyl optionally protected with a protecting group, nitro andsulfonyl;

-   n represents an integer of 0 to 3;-   R¹ represents lower alkyl;-   R² and R³ may be the same or different and represent at least one    species selected from the group consisting of hydrogen and lower    alkyl; and-   * represents asymmetric carbon,-   or a salt thereof, to produce an optically active aminoalcohol    compound represented by the following general formula (2):

wherein X, n, R¹, R², R³ and * have the same definitions as above, andhaving the desired optical activity.

-   (37) A method for production of an optically active aminoalcohol,    wherein a transformant according to (34) is reacted with an    enantiomeric mixture of an α-aminoketone compound represented by the    following general formula (1):

wherein X may be the same or different and represents at least onespecies selected from the group consisting of halogen, lower alkyl,hydroxyl optionally protected with a protecting group, nitro andsulfonyl;

-   n represents an integer of 0 to 3;-   R¹ represents lower alkyl;-   R² and R³ may be the same or different and represent at least one    species selected from the group consisting of hydrogen and lower    alkyl; and-   * represents asymmetric carbon,-   or a salt thereof, to produce an optically active aminoalcohol    compound represented by the following general formula (2):

wherein X, n, R¹, R², R³ and * have the same definitions as above, andhaving the desired optical activity.

-   (38) A production method for an optically active aminoalcohol    according to (37), wherein the production method for the optically    active aminoalcohol is carried out with further addition of a    compound represented by the following general formula (3):

wherein A represents the following formula (Y) or (Z):

wherein R⁴ represents hydrogen, optionally substituted C1-3 alkyl, aC5-10 hydrocarbon ring which is bonded to R⁸ or a 5- to 8-memberedheterocyclic skeleton containing 1-3 heteroatoms which is bonded to R⁸,

wherein R⁵ represents hydrogen, C1-3 alkyl or a 5- to 8-memberedheterocyclic skeleton containing 1-3 heteroatoms which is bonded to R⁶or R⁹;

-   R⁶ represents hydrogen, optionally substituted C1-3 alkyl, a C5-10    hydrocarbon ring which is bonded to R⁸ or a 5- to 8-membered    heterocyclic skeleton containing 1-3 heteroatoms which is bonded to    R⁵ or R⁹;-   R⁷ represents hydrogen or optionally substituted C1-6 alkyl;-   R⁸ represents hydrogen, carboxyl, optionally substituted C1-6 alkyl,    a 5- to 8-membered heterocyclic skeleton containing 1-3 heteroatoms    which is bonded to R⁴ or a C5-10 hydrocarbon ring which is bonded to    R⁶;-   R⁹ represents hydrogen, optionally substituted C1-6 alkyl,    optionally substituted C1-6 alkyloxycarbonyl, optionally substituted    acyl or a 5- to 8-membered heterocyclic skeleton containing 1-3    heteroatoms which is bonded to R⁵ or R⁶; and-   R¹⁰ represents hydrogen or optionally substituted C1-6 alkyl, or a    pharmaceutically acceptable salt or solvate thereof, for production    of an optically active aminoalcohol.-   (39) A shuttle vector according to (27), having a nucleotide    sequence selected from the group consisting of the nucleotide    sequences set forth as SEQ ID NO: 89 to SEQ ID NO: 100.

EFFECT OF THE INVENTION

The plasmids of the invention are novel plasmids unknown to the priorart, and are valuable as vectors for host-vector systems belonging tothe industrially useful the genus Rhodococcus. They are of particularutility in the creation of recombinant microbes capable of industrialasymmetric reduction of aminoketones. An example of asymmetric reductionof an aminoketone to which such microbes may contribute is a reactionfor production of d-(1S, 2S)-pseudoephedrine from1-2-methylamino-1-phenyl-1-propanone.

The plasmids of the invention can coexist in single Rhodococcus cell andtherefore can be used not only alone for their replicating function, butalso as compatible plasmids. That is, by inserting different protein(for example, enzyme) genes into the different plasmids, it is possibleto express the proteins simultaneously in the same cell.

The shuttle vectors of the invention are useful for creation ofindustrially useful recombinant microbes of the genus Rhodococcus andEscherichia coli.

The nucleotide sequence data relating to DNA replication obtained fromthe plasmids of the invention may serve as the basis for construction ofthe aforementioned shuttle vectors, and specifically they provide DNAfragments as constituent elements of the vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction enzyme cleavage map of plasmid pRET1100.

FIG. 2 is a restriction enzyme cleavage map of plasmid pRET1000.

FIG. 3 is a summary illustration for construction of shuttle vectorpRET1101.

FIG. 4 is a summary illustration for construction of shuttle vectorpRET1102.

FIG. 5 is a summary illustration for construction of shuttle vectorpRET1103.

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the invention will now be explained.

The first plasmid of the invention is a plasmid isolated from the genusRhodococcus, or a derivative thereof. Specifically, it may be isolatedfrom, for example, Rhodococcus erythropolis IAM1400, IAM1503, JCM2893and JCM2894 strains, has a size of approximately 5.4 kbp and is acircular plasmid cleavable by the restriction enzymes shown in Table 1.The plasmids isolated from each of these strains are designated aspRET1100, pRET1300, pRET1500 and pRET1700, respectively. Plasmids of theinvention may be prepared from these sample strains by publicly knownmethods (for example, boiling, alkali dissolution, cesium chloridedensity gradient ultracentrifugation: Lab Manual Idenshi Kogaku, 3rdEdition, Chapter 10, pp. 55-59, Maruzen).

TABLE 1 Restriction Number of Fragment sizes enzyme cleavage sites (kbp)BamH I 2 0.4, 5.0 EcoR I 2 0.3, 5.1 Kpn I 1 5.4 Pvu II 1 5.4 Sac I 1 5.4Sma I 1 5.4

FIG. 1 shows a restriction enzyme cleavage map for pRET1100. Thisplasmid was sequenced by a publicly known method (using a fluorescentautomatic sequencer, for example) and its full nucleotide sequence wasrevealed to be 5444 bp set forth as SEQ ID NO: 73 of the SequenceListing.

The second plasmid of the invention is also a plasmid isolated from thegenus Rhodococcus, or its derivative. Specifically, it may be isolatedfrom, for example, Rhodococcus rhodnii JCM3203, has a size ofapproximately 5.8 kbp and is a circular plasmid cleavable by therestriction enzymes shown in Table 2. This plasmid is designated aspRET1000.

TABLE 2 Restriction Number of enzyme cleavage sites Fragment sizes (kbp)BamH I 2 2.0, 3.8 Pvu II 4 0.1, 1.4, 2.0, 2.3 Sac I 3 0.9, 1.0, 3.9 SmaI 4 0.1, 1.2, 1.6, 2.9

FIG. 2 shows a restriction enzyme cleavage map for pRET1000. Thisplasmid was also sequenced by a publicly known method and its fullnucleotide sequence was revealed to be 5813 bp set forth as SEQ ID NO:74 of the Sequence Listing.

The plasmids of the invention (natural- or wild-types) are circularplasmids that can also be defined by the restriction enzyme cleavagepatterns shown in Tables 1 and 2. Thus, the present inventionencompasses the following two types of circular plasmids.

-   -   (1) A circular plasmid derived from a Rhodococcus strain,        characterized by having a size of approximately 5.4 kbp and        possessing the following restriction enzyme cleavage sites: BamH        I:2, EcoR I:2, Kpn I:1, Pvu II:1, Sac I:1 and Sma I:1.    -   (2) A circular plasmid derived from a Rhodococcus strain,        characterized by having a size of approximately 5.8 kbp and        possessing the following restriction enzyme cleavage sites: BamH        I:2, Pvu II:4, Sac I:3 and Sma I:4.

As a result of analysis of the nucleotide sequences of plasmids pRET1100and pRET1000 (i.e., SEQ ID NO: 73 and SEQ ID NO: 74), there is predictedthe existence of a group of nucleotide sequences (open reading frames,hereinafter “orf”) coding for proteins for DNA replication or otherfunctions.

In the relevant technical field, “DNA replication” refers to using DNAitself as template to form two double-stranded DNA molecules exactlyidentical to existing double-stranded DNA (parent DNA). The replicationmechanism consists of three stages: initiation from the starting pointof replication (replication origin), DNA chain elongation andtermination. During replication, a portion of the DNA double strand isunraveled and new DNA strands are synthesized complementary to eachsingle strand. The double strand is unraveled by DNA helicase and helixdestabilizing proteins (also known as single-strand DNA-bindingprotein), and the unraveled portion is referred to as the replicationfork. The template DNA in the direction from 3′ to 5′ toward thereplication fork is the “leading strand”, and the one in the directionfrom 5′ to 3′ is the “lagging strand”. DNA polymerase extends the DNAstrand in the direction from 5′ to 3′. Therefore when the leading strandis the template, DNA is synthesized in the direction of the replicationfork. However when the opposite lagging strand is the template, the DNAstrand must be extended in the opposite direction from the replicationfork. Consequently, replication of the lagging strand is accomplished infragments of about 200 bases, known as Okazaki fragments. Everyapproximately 200 bases, RNA primer is used with DNA as template tosynthesize 10 bases of RNA in the direction from 5′ to 3′. From this RNAas primer, DNA polymerase synthesizes a DNA strand in the direction from5′ to 3′ on the lagging strand as template. The replicated DNA fragmentof approximately 200 bases then binds to the single-stranded DNA fromwhich RNA is removed. In this replication mechanism, several proteinsincluding DNA helicase and helix-destabilizing protein work together toform the replicating machinery. Other proteins involved include DNAtopoisomerase (which prevents twisting during the DNA replication),replication initiation proteins and replication termination proteins.The DNA replication mechanism is described in detail in, for example,“Saibou no Bunshiseibutsugaku [Molecular Biology of the Cell]”, 3rdEdition, translated by Keiko Nakamura et al., pp. 251-262, Kyoikusha,1996.

Upon analysis of the nucleotide sequences of the plasmids pRET1100 andpRET1000, they were found to include sequences of AT-rich homologous oranalogous repeats and a sequence thought to have a DNA secondarystructure, i.e. a nucleotide sequence predicted to be a DNA replicationregion (a nucleotide sequence region recognized by proteins involved inDNA replication or a region including the DNA replication origin), inthe vicinity of the aforementioned orf relating to DNA replication.

DNA replication requires a DNA replication region and a region codingfor a protein involved in DNA replication (hereinafter referred to as“DNA replication-related protein”). According to the present inventionit is possible to obtain data relating to the nucleotide sequences ofthese regions for both plasmids pRET1100 and pRET1000.

First, the nucleotide sequences set forth as SEQ ID NO: 35-37 wereidentified as DNA replication regions for plasmid pRET1100. As regionscoding for proteins related to DNA replication there were identified thenucleotide sequences set forth as SEQ ID NO: 1-3 (orf1), the nucleotidesequence set forth as SEQ ID NO: 4 (orf2), the nucleotide sequences setforth as SEQ ID NO: 5-16 (orf3), the nucleotide sequences set forth asSEQ ID NO: 17-21 (orf4), the nucleotide sequences set forth as SEQ IDNO: 22-26 (orf5), the nucleotide sequence set forth as SEQ ID NO: 27 or28 (orf6), the nucleotide sequence set forth as SEQ ID NO: 29 or 30(orf7), the nucleotide sequence set forth as SEQ ID NO: 31 or 32 (orf8),and the nucleotide sequence set forth as SEQ ID NO: 33 or 34 (orf9).

Construction of a plasmid capable of DNA replication from pRET1100requires that the recombinant plasmid have at least one DNA replicationregion and at least one coding region (orf) for a DNAreplication-related protein. Thus, the (recombinant) plasmids of theinvention are characterized by comprising at least one DNA replicationregion and at least one coding region for a DNA replication-relatedprotein. The coding region for a DNA replication-related proteinpreferably has a nucleotide sequence selected from the group consistingof the nucleotide sequences set forth as SEQ ID NO: 1, 4, 14, 17 and 22.

The region of the nucleotide sequence set forth as SEQ ID NO: 76 hasbeen suggested as a promoter involved in expression ofreplication-related proteins, and the plasmids of the inventionpreferably comprise a promoter region having the nucleotide sequence setforth as SEQ ID NO: 76.

For plasmid construction, the DNA fragments are appropriately selectedbased on the aforementioned nucleotide sequence data. The presentinvention also encompasses derivatives or functional (DNA-replicating)fragments of the plasmids.

Next, the nucleotide sequences set forth as SEQ ID NO: 70-72 wereidentified as DNA replication regions for plasmid pRET1000. As regionscoding for proteins related to DNA replication there were identified thenucleotide sequences set forth as SEQ ID NO: 38-41 (orf10), thenucleotide sequence set forth as SEQ ID NO: 42 or 43 (orf11), thenucleotide sequence set forth as SEQ ID NO: 44 (orf12), the nucleotidesequence set forth as SEQ ID NO: 45 or 46 (orf13), the nucleotidesequences set forth as SEQ ID NO: 48-50 (orf14), the nucleotide sequenceset forth as SEQ ID NO: 51 or 52 (orf15), the nucleotide sequence setforth as SEQ ID NO: 53 or 54 (orf16), the nucleotide sequence set forthas SEQ ID NO: 55 (orf17), the nucleotide sequences set forth as SEQ IDNO: 56-60 (orf18), the nucleotide sequence set forth as SEQ ID NO: 61(orf19), the nucleotide sequence set forth as SEQ ID NO: 62 (orf20), andthe nucleotide sequences set forth as SEQ ID NO: 63-69 (orf21).

Construction of a plasmid capable of DNA replication from pRET1000requires that the recombinant plasmid have at least one DNA replicationregion and at least one coding region (orf) for a DNAreplication-related protein. Thus, the (recombinant) plasmids of theinvention are characterized by comprising at least one DNA replicationregion and at least one coding region for a DNA replication-relatedprotein. The coding region for a DNA replication-related proteinpreferably has a nucleotide sequence selected from the group consistingof the nucleotide sequences set forth as SEQ ID NO: 40, 42, 44, 45, 53,55, 56, 61, 62 and 69.

The regions with the nucleotide sequences set forth as SEQ ID NO: 67 and47 are homologous with mobilization proteins, and have been implicatedin mobilization. The region with the nucleotide sequence set forth asSEQ ID NO: 75 has been implicated in gene expression of mobilizationprotein and suggested as a recognition site for an expressed protein.Thus, the plasmids of the invention preferably include mobilizationprotein regions having the nucleotide sequences set forth as SEQ ID NO:67 and 47, or include a region involved in mobilization having thenucleotide sequence set forth as SEQ ID NO: 75.

For plasmid construction, the DNA fragments are appropriately selectedbased on the aforementioned nucleotide sequence data. The presentinvention also encompasses derivatives or functional (DNA-replicating)fragments of the plasmids.

The plasmids or DNA fragments of the invention may also containnucleotide sequences with a substitution, deletion or insertion of oneor a plurality of nucleotides in a DNA replication region, DNAreplication-related protein coding region, promoter region, mobilizationprotein region or mobilization-related region, or a portion thereof, solong as the function of each region is not impaired.

The shuttle vectors of the invention may be any which comprise a plasmidor DNA fragment having a DNA replication region, DNA replication-relatedprotein coding region, promoter region, mobilization protein region ormobilization-related region, and a DNA region that is replicable in E.coli, and which are replicable in the genus Rhodococcus and E. coli,such as those having the nucleotide sequences set forth as SEQ ID NO: 89to 100. The shuttle vectors of the invention may also have nucleotidesequences with one or a plurality of nucleotide substitutions, deletionsor insertions in the aforementioned nucleotide sequences, so long asthey are replicable in the genus Rhodococcus and E. coli.

The “plurality” referred to above will differ depending on the type ofregion, and specifically may be 2-1100, preferably 2-800, morepreferably 2-300, even more preferably 2-100, yet more preferably 2-20and most preferably 2-10.

As a plasmid or DNA fragment having substantially the same nucleotidesequence as the aforementioned DNA replication region, DNAreplication-related protein coding region, promoter region, mobilizationprotein region or mobilization-related region, or a portion thereof,there may be mentioned specifically, a nucleotide sequence whichhybridizes with a DNA replication region, DNA replication-relatedprotein coding region, promoter region, mobilization protein region ormobilization-related region, or a portion thereof, under stringentconditions. Here “stringent conditions” are conditions under whichspecific hybrids are formed and non-specific hybrids are not formed.While it is difficult to precisely quantify the conditions, one exampleis a set of conditions that permit hybridization of DNA with highhomology, such as 80% or greater, preferably 90% or greater or morepreferably 95% or greater homology, while not permitting hybridizationof DNA with lower homology. More specifically, there may be mentionedhybridization conditions with ordinary Southern hybridization washing at60° C., 1×SSC, 0.1% SDS or preferably Southern hybridization washing at0.1×SSC, 0.1% SDS corresponding salt concentration. When a DNA fragmentwith a length of approximately 300 bp is used as a portion of the DNAreplication region, DNA replication-related protein coding region,promoter region, mobilization protein region or mobilization-relatedregion, the hybridization washing conditions may be 50° C., 2×SSC, 0.1%SDS.

The aforementioned plasmid or DNA fragment having substantially the samenucleotide sequence as the aforementioned DNA replication region, DNAreplication-related protein coding region, promoter region, mobilizationprotein region or mobilization-related region, or a portion thereof, maybe obtained by for example, modification of a DNA replication region,DNA replication-related protein coding region, promoter region,mobilization protein region or mobilization-related region, or a portionthereof, by site-directed mutagenesis so as to have a substitution,deletion or insertion of nucleotides at a specific site. Such modifiedDNA may also be obtained by mutation treatment known in the prior art.As mutation treatments there may be mentioned methods of in vitrotreatment of DNA including a DNA replication region, DNAreplication-related protein coding region, promoter region, mobilizationprotein region or mobilization-related region, or a portion thereof,with hydroxylamine or the like, and methods of treating a microbepossessing the DNA above, such as the genus Escherichia, withultraviolet rays or with a mutagenic agent ordinarily used formutagenesis such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or EMS.

Nucleotide substitutions, deletions or insertions as mentioned aboveinclude those found in naturally occurring mutants or variants due todifferences in Rhodococcus strains.

A shuttle vector of the invention includes a DNA fragment (A) as theaforementioned plasmid or portion thereof, and a DNA region (B) which isreplicable in E. coli. In some cases it is preferred for the shuttlevector to comprise a DNA region including a drug resistance gene. In therelevant technical field, a “shuttle vector” is a vector which comprisesthe DNA replication mechanism for two different cell types, andpreferably also a drug resistance gene or the like as a selectivemarker, allowing its auto-replication in the two different cell-types.The DNA fragment (A) as the aforementioned plasmid or portion thereof isa DNA region that is replicable in the genus Rhodococcus. The DNA region(B) which is replicable in E. coli may be a full plasmid or a portionthereof, so long as it can be replicated and amplified in E. coli. Assuch DNA regions that are replicable in E. coli there may be used, forexample, the plasmids pUC18, pHSG299 and pHSG398.

When the shuttle vector of the invention includes a drug resistancegene, the preferred ones are ampicillin resistance gene, kanamycinresistance gene and chloramphenol resistance gene, but there are noparticular restrictions on the manner of drug so long as the gene isexpressed in the genus Rhodococcus and E. coli as hosts and confers drugresistance to the host cells, in order to allow verification of thepresence of plasmids in the two genera based on resistance to the drug.Also, a plurality of such drug resistance genes may be used incombination.

The shuttle vector preferably contains multiple cloning sites(multicloning sites), and the cloning sites and drug resistance gene maybe induced from, for example, an E. coli plasmid. That is, a publiclyknown E. coli plasmid such as one listed above may be cleaved with anappropriate restriction endonuclease and a DNA region containing thecloning sites and drug resistance gene constructed and ligated withanother DNA fragment (a DNA region which is replicable in the genusRhodococcus).

As an illustration, outline of shuttle vector constructions is shown inFIGS. 3 to 5. The shuttle vectors may be constructed by treating theaforementioned plasmids and E. coli plasmids With suitable restrictionendonucleases and then ligating them. In this manner, the presentinventors constructed 18 shuttle vectors (Table 5) using the Rhodococcusplasmids pRET1000, pRET1100 or pRET1200, and the E. coli plasmids pUC18,pHSG299 or pHSG398.

The shuttle vectors of the invention are replicable in the genusRhodococcus and E. coli as hosts, and are industrially useful. TheRhodococcus and E. coli strains transformed by the shuttle vectors ofthe invention, as well as other microbial transformants, are useful inthis way and such transformants are also encompassed by the scope of theinvention.

A vector of the invention is characterized by being constructed using ashuttle vector of the invention. Specifically, it is a vector havingtarget DNA inserted therein which is to be introduced into the shuttlevector of the invention. The DNA to be introduced and the shuttle vectorof the invention are treated with appropriate restriction endonucleasesand then ligated them to construct the vector. The vector may then beused to obtain transformants having the desired DNA transferred therein.

As examples of DNA to be inserted there may be mentioned aminoketoneasymmetric reductase genes and coenzyme-regenerating system enzymegenes. Aminoketone asymmetric reductase genes are genes coding foraminoketone asymmetric reductases as described in WO02/070714, and morespecifically, DNA coding for a protein comprising the amino acidsequence set forth as SEQ ID NO: 78 (aminoketone asymmetric reductasederived from R. erythropolis MAK-34), and particularly DNA comprisingthe nucleotide sequence set forth as SEQ ID NO: 79. The entirety of thecontent described in WO02/070714 is incorporated herein by reference.

An aminoketone asymmetric reductase is any having the propertiesdescribed in WO02/070714, and includes a protein having the amino acidsequence set forth as SEQ ID NO: 78 of the Sequence Listing, as well asproteins having amino acid sequences obtained by deletion, insertion,substitution or addition of one or more amino acids in theaforementioned amino acid sequence, and exhibiting aminoketoneasymmetric reduction activity. Aminoketone asymmetric reduction activityis activity of producing an optically active aminoalcohol represented bygeneral formula (2) above using an α-aminoketone represented by generalformula (1) above as the substrate.

There are no particular restrictions on the methods of deletion,insertion, substitution and addition, and any publicly known methods maybe employed. For example, there may be mentioned the methods describedin “Zoku Seikagaku Jikken Kouza 1, Idenshi Kenkyuhou II”, edited by theJapanese Biochemical Society, p 105 (Hirose, S.), Tokyo Kagaku Dojin(1986); “Shin Seikagaku Jikken Kouza 2, Kakusan III (Recombinant DNATechnology)”, edited by the Japanese Biochemical Society, p. 233(Hirose, S.), Tokyo Kagaku Dojin (1992); R. Wu, L. Grossman ed.,“Methods in Enzymology”, Vol. 154, p. 350 & p. 367, Academic Press, NewYork (1987); R. Wu, L. Grossman, ed., “Methods in Enzymology”, Vol. 100,p. 457 & p. 468, Academic Press, New York (1983); J. A. Wells et al.,“Gene”, Vol. 34, p. 315 (1985);. T. Grundstroem et al., “Nucleic AcidsRes”, Vol. 13, p. 3305 (1985); J. Taylor et al., “Nucleic Acids Res.”,Vol. 13, p. 8765 (1985); R. Wu, ed., “Methods in Enzymology”, Vol. 155,p. 568, Academic Press, New York (19.87); and A. R. Oliphant et al.,“Gene”, Vol. 44, p. 177 (1986). As specific examples, there may bementioned the site-directed mutagenesis method (site-specificmutagenesis method) utilizing synthetic oligonucleotides, the Kunkelmethod, the dNTP[αS] method (Eckstein method), and the region-directedmutagenesis method using sulfurous acid or nitrous acid.

Sugar chains are attached to the majority of proteins, and substitutionof one or a plurality of amino acids can modify the attachment of sugarchains. Thus, the aminoketone asymmetric reductases of the inventionalso include proteins having the amino acid sequence set forth as SEQ IDNO: 78 of the Sequence Listing and having modifications of sugar chains,so long as they exhibit the aforementioned aminoketone asymmetricreduction activity.

The aminoketone asymmetric reductases of the invention may also havemodifications of their amino acid residues by chemical methods, or theirderivatives may be enhanced by modification or partial degradation usingpeptidase enzymes such as pepsin, chymotrypsin, papain, bromelain,endopeptidase and exopeptidase.

When the aminoketone asymmetric reductases of the invention are producedby a gene recombinant method, a fusion protein may be expressed and thenconverted or processed into a protein having biological activity whichis substantially equivalent to a natural aminoketone asymmetricreductase either in vivo or ex vivo. In this case, a fusion productionmethod ordinarily employed for genetic engineering may be used, and thefusion protein may be purified by affinity chromatography or the like,utilizing the fused portion thereof. Modification and enhancement ofprotein structures may be carried out with reference to “Shin SeikagakuJikken Kouza 1, Tanpakushitsu VII, Tanpakushitsu Kogaku”, edited by theJapanese Biochemical Society, Tokyo Kagaku Dojin (1993), by the methodsdescribed therein, the methods described in literature cited therein, ormethods which are essentially equivalent thereto.

The aminoketone asymmetric reductase of the invention may also differfrom naturally occurring forms in the identities of one or more of theamino acid residues or in the positions of one or more of the amino acidresidues. The present invention also encompasses deletion analogues withdeletion of one or more (for example, 1-80, preferably 1-60, morepreferably 1-40, even more preferably 1-20 and especially 1-10) aminoacid residues, substitution analogues with substitution of one or more(for example, 1-80, preferably 1-60, more preferably 1-40, even morepreferably 1-20 and especially 1-10) amino acid residues or additionanalogues with addition of one or more (for example, 1-80, preferably1-60, more preferably 1-40, even more preferably 1-20 and especially1-10) amino acid residues peculiar to natural aminoketone asymmetricreductases. Also encompassed are enzymes having the domain structurecharacteristic of natural aminoketone asymmetric reductases. There mayalso be mentioned isomers of the aminoketone asymmetric reductases.

So long as the domain structure characteristic of natural aminoketoneasymmetric reductases is maintained, all mutants above are alsoencompassed among the aminoketone asymmetric reductases of theinvention. In addition, it is assumed that enzymes having a primarystructural conformation substantially equivalent to natural aminoketoneasymmetric reductases of the invention, or a portion thereof, as well asenzymes having biological activity substantially equivalent to naturalaminoketone asymmetric reductases, may also be included. Naturallyoccurring mutants may also be mentioned. The aminoketone asymmetricreductases of the invention may be separated and purified in the mannerexplained below. The present invention encompasses DNA fragments codingfor the aforementioned polypeptides, polypeptides of aminoketoneasymmetric reductases having all or some of the natural features, andDNA fragments coding for analogues or derivatives thereof. Thenucleotides of the aminoketone asymmetric reductases may be modified(for example, with addition, deletions or substitutions), and suchmodified forms are also encompassed by the invention.

An aminoketone asymmetric reductase gene according to the invention is anucleic acid coding for any of the aforementioned aminoketone asymmetricreductases. As representative examples there may be mentioned nucleicacid coding for a protein having the amino acid sequence set forth asSEQ ID NO: 78 of the Sequence Listing, and especially nucleic acidhaving the nucleotide sequence set forth as SEQ ID NO: 79, but sinceseveral nucleotide sequences (codons) can code for each amino acid,there exist numerous nucleic acids coding for a protein having the aminoacid sequence set forth as SEQ ID NO: 78. Thus, all such nucleic acidsare also encompassed among the aminoketone asymmetric reductase genes ofthe invention. Here, “coding for a protein” means that, when the DNAconsists of two strands, one of the two complementary strands has anucleotide sequence coding for the protein, and therefore the nucleicacids of the invention include nucleic acids comprising nucleotidesequences directly coding for the amino acid sequence set forth as SEQID NO: 78 and nucleic acids comprising nucleotide sequences which arecomplementary thereto. In addition, the aminoketone asymmetric reductasegenes of the invention may be nucleic acids which hybridize with nucleicacid comprising a nucleotide sequence complementary to SEQ ID NO: 79under stringent conditions, and which code for proteins with aminoketoneasymmetric reduction activity. Here, “stringent conditions” has the samedefinition as explained above.

The coenzyme-regenerating system enzyme gene may be one for variousdehydrogenases, specifically, glucose dehydrogenase, glucose-6-phosphatedehydrogenase, aldehyde dehydrogenases, alcohol dehydrogenases, organicacid dehydrogenases and amino acid dehydrogenases. More specifically,there may be suitably used acetaldehyde dehydrogenase, ethanoldehydrogenase, propanol dehydrogenase, glycerol dehydrogenase, formatedehydrogenase, acetate dehydrogenase, butyrate dehydrogenase, lactatedehydrogenase, maleate dehydrogenase and glutamate dehydrogenase.

A transformant according to the invention is characterized by comprisingthe aforementioned vector. The transformant is obtained by introducingthe vector into host cells. The vector introduction method may be apublicly known method, such as the calcium phosphate method,lipofection, electroporation, microinjection or the like.

For example, a transformant of the invention comprising a vector havingan aminoketone asymmetric reductase gene inserted therein hasaminoketone asymmetric reduction activity, and may be applied for anaminoketone asymmetric reductase production method or optically activeaminoalcohol production method as described below.

The method for production of an aminoketone asymmetric reductase of theinvention is characterized by comprising a culturing step in whichtransformants containing a vector having an aminoketone asymmetricreductase gene inserted therein are cultured in medium which allowsgrowth of the transformants, and a purification step in which theaminoketone asymmetric reductase is purified from the transformantsobtained in the culturing step.

The method for culturing may be a publicly known method with noparticular restrictions so long as it permits growth of the cells used,and ordinarily a liquid medium containing a carbon source, nitrogensource and other nutrients is used. As carbon sources for the mediumthere may be used any of those that can be utilized by the cells.Specifically, there may be mentioned sugars such as glucose, fructose,sucrose, dextrin, starch and sorbitol, alcohols such as methanol,ethanol and glycerol, organic acids such as fumaric acid, citric acid,acetic acid and propionic acid, and their salts, hydrocarbons such asparaffin, and mixtures thereof. As nitrogen sources there may be usedany of those that can be utilized by the cells. Specifically, there maybe mentioned ammonium salts of inorganic acids such as ammoniumchloride, ammonium sulfate and ammonium phosphate; ammonium salts oforganic acids such as ammonium fumarate and ammonium citrate; nitricacid salts such as sodium nitrate and potassium nitrate; and inorganicor organic nitrogenous compounds such as meat extract, yeast extract,malt extract and peptone, as well as mixtures thereof. The medium mayalso contain appropriately added nutrient sources ordinarily used forculturing, such as inorganic salts, trace metal salts and vitamins; Whennecessary, there may also be added to the medium substances that promotecell growth and buffering substances effective for maintaining the pH ofthe medium.

The culturing of the cells may be carried out under conditions suitablefor growth. Specifically, the medium pH may be 3-10, preferably 4-9, andthe temperature may be 0-50° C., preferably 20-40° C. The cell culturingmay be conducted either under aerobic or anaerobic conditions. Theculturing time is preferably 10-150 hours, but should be appropriatelydetermined for the type of cells used.

The culture solution of the cells cultured in the manner described aboveis filtered or centrifuged and the cells are rinsed with water or buffersolution. The rinsed cells are suspended in a suitable amount of buffersolution for disruption of the cells. The method of disruption is notparticularly restricted but as examples there may be mentionedmechanical disruption with a mortar, Dynomill, French press, ultrasoniccell disrupter or the like. The aminoketone asymmetric reductase in thecell-free extract obtained by filtration or centrifugation of the solidmatter from the cell disruptate is recovered by an ordinary enzymeisolating method.

There are no particular restrictions on the method for isolation of theenzyme and any publicly known method may be employed, but as examplesthere may be mentioned purification by salting out such as ammoniumsulfate precipitation; gel filtration methods using Sephadex and thelike; ion-exchange chromatography methods using carriers withdiethylaminoethyl groups or carboxymethyl groups; hydrophobicchromatography using carriers with hydrophobic groups such as butyl,octyl and phenyl; dye gel chromatography methods; electrophoresismethods; dialysis; ultrafiltration methods; affinity chromatographymethods; high performance liquid chromatography methods and the like.

The enzyme may also be used as an immobilized enzyme. There are noparticular restrictions on the method and any publicly known method maybe employed, among which there may be mentioned immobilization of theenzyme or the enzyme-producing cells, and the immobilization may beaccomplished by a carrier bonding method such as a covalent bondingmethod or adsorption method, a crosslinking method, entrapment method orthe like. A condensing agent such as glutaraldehyde, hexamethylenediisocyanate or hexamethylene diisothiocyanate may also be used ifnecessary. Other immobilizing methods include: a monomer method in whicha monomer is gelled by polymerizing reaction; a prepolymer method inwhich molecules larger than monomers are polymerized; a polymer methodin which a polymer is gelled; immobilization using polyacrylamide;immobilization using natural polymers such as alginic acid, collagen,gelatin, agar and κ-carrageenan; and immobilization using syntheticpolymers such as photosetting resins and urethane polymers.

The enzyme purified in this manner is judged as having been adequatelypurified if a single band is confirmed in electrophoresis (SDS-PAGE,etc.).

A method for production of an optically active aminoalcohol according tothe invention is characterized to produce an optically activeaminoalcohol compound represented by the following general formula (2),which compound exhibits the desired optical. activity, by reacting anaminoketone asymmetric reductase obtained by the production method ofthe invention with an enantiomeric mixture of an α-aminoketone compoundrepresented by the following general formula (1) or a salt thereof.

wherein X may be the same or different and represents at least onespecies selected from the group consisting of halogen, lower alkyl,hydroxyl optionally protected with a protecting group, nitro andsulfonyl;

-   n represents an integer of 0 to 3;-   R¹ represents lower alkyl;-   R² and R³ may be the same or different and represent at least one    species selected from the group consisting of hydrogen and lower    alkyl; and-   * represents asymmetric carbon.

wherein X, n, R¹, R², R³ and * have the same definitions as above.

First, the α-aminoketone compound represented by general formula (1)according to the invention will be explained.

The substituent X is as follows. As the aforementioned halogen there maybe mentioned fluorine, chlorine, bromine and iodine.

As lower alkyl there are preferred C1-6 alkyl, among which there may bementioned methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl,t-butyl, pentyl, isopentyl, hexyl and the like. These may havestraight-chain or branched structures. As substituents they may containhalogens such as fluorine or chlorine, or hydroxyl, alkyl, amino, alkoxyand the like.

As protecting groups for hydroxyl optionally protected with a protectinggroup there may be mentioned groups that can be removed by treatmentwith water, groups that can be removed by acid or weak base treatment,groups that an be removed by hydrogenation or groups that can be removedwith Lewis acid catalysts and thiourea, and such protecting groupsinclude optionally substituted acyl, optionally substituted silyl,alkoxyalkyl, optionally substituted lower alkyl, benzyl,p-methoxybenzyl, 2,2,2-trichloroethoxycarbonyl, allyloxycarbonyl, trityland the like.

The aforementioned acyl groups include acetyl, chloroacetyl,dichloroacetyl, pivaloyl, benzoyl, p-nitrobenzoyl and the like. They maycontain hydroxyl, alkyl, alkoxy, nitro, halogen and the like assubstituents. The aforementioned silyl groups include trimethylsilyl,t-butyldimethylsilyl, triarylsilyl and the like. They may contain alkyl,aryl, hydroxyl, alkoxy, nitro, halogen and the like as substituents. Theaforementioned alkoxyalkyl groups include methoxymethyl,2-methoxyethoxymethyl and the like. The aforementioned lower alkylinclude C1-6 alkyl, among which there may be mentioned methyl, ethyl,propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, isopentyl,hexyl and the like. These may have straight-chain or branchedstructures. As substituents they may contain halogen such as fluorine orchlorine, or hydroxyl, alkyl, amino, alkoxy and the like.

X may be nitro or sulfonyl, and specifically there may be mentionedmethylsulfonyl and the like.

The number “n” for X is an integer of 0-3, and is preferably 0.

R¹ in general formula (1) above represents lower alkyl. As lower alkylthere are preferred C1-6 alkyl, among which there may be mentionedmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl,pentyl, isopentyl, hexyl and the like. These may have straight-chain orbranched structures.

Each of R² and R³ represent hydrogen or lower alkyl. The lower alkylinclude C1-6 alkyl, among which there may be mentioned methyl, ethyl,propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, isopentyl,hexyl and the like. These may have straight-chain or branchedstructures.

As salts of the aforementioned α-aminoketone compounds there may bementioned salts of inorganic acids such as hydrochloride, sulfate,nitrate, phosphate and carbonate, and salts of organic acids such asacetic acid and citric acid.

The α-aminoketone can be easily synthesized by halogenation (forexample, bromination) of the α-carbon of a corresponding 1-phenylketonederivative, followed by replacement of the halogen such as bromine withan amine (Ger. (East), 11, 332, Mar. 12, 1956).

The optically active aminoalcohol represented by general formula (2)above according to the invention will now be explained. In generalformula (2), X, n, R¹, R², R³ and * have the same definitions as ingeneral formula (1) above. As β-aminoalcohols having the desired opticalactivity there may be mentioned (1S, 2S)aminoalcohols. As specificexamples of (1S, 2S)aminoalcohols there may be mentionedd-threo-2-methylamino-1-phenylpropanol (d-pseudoephedrine),d-threo-2-dimethylamino-1-phenylpropanol (d-methylpseudoephedrine), (1S,2S)-α-(1-aminoethyl)-benzyl alcohol (d-norpseudoephedrine), (1S,2S)-1-(p-hydroxyphenyl)-2-methylamino-1-propanol, (1S,2S)-α-(1-aminoethyl)-2,5-dimethoxy-benzyl alcohol, (1S,2S)-1-(m-hydroxyphenyl)-2-amino-1-propanol, (1S,2S)-1-(p-hydroxyphenyl)-2-amino-1-propanol, (1S,2S)-1-phenyl-2-ethylamino-1-propanol, (1S,2S)-1-phenyl-2-amino-1-butanol, (1S,2S)-1-phenyl-2-methylamino-1-butanol and the like.

The conditions for reaction of the aminoketone asymmetric reductase arenot particularly restricted so long as an optically active aminoalcoholrepresented by general formula (2) having the desired optical activityis produced, but since the enzyme optimum pH is 8.1 and the optimumtemperature is 55° C., the reaction is preferably carried out underconditions of pH 7-9 and 30-65° C. temperature.

A method for production of an optically active aminoalcohol according tothe invention is also characterized to produce an optically activeaminoalcohol compound represented by the following general formula (2),which compound exhibits the desired optical activity, by reacting atransformant of the invention with an enantiomeric mixture of anα-aminoketone compound represented by the following general formula (1)or a salt thereof.

As the reaction conditions for the reaction described above, forexample, the transformants shake cultured in liquid medium may becollected, an aqueous aminoketone solution (0.1-10% concentration) addedto the obtained cells, and reaction conducted at a temperature of 20-40°C. for a period of several hours to one day while regulating the pH tobetween 6-8. Upon completion of the reaction, the cells may be separatedand the product in the reaction solution isolated to obtain an opticallyactive aminoalcohol. The reaction may be conducted in the same mannerfor treated transformant cells (dry cells or immobilized cells) or theenzyme or immobilized enzyme obtained from the transformants.

In the production method for an optically active aminoalcohol of theinvention, the reaction may be carried out with further addition of acompound represented by the following general formula (3) or apharmaceutically acceptable salt or solvate thereof, for more efficientproduction of the optically active aminoalcohol.

(wherein A represents the following formula (Y) or (Z))

(wherein R⁴ represents hydrogen, optionally substituted C1-3 alkyl, aC5-10 hydrocarbon ring which is bonded to R⁸ or a 5- to 8-memberedheterocyclic skeleton containing 1-3 heteroatoms which is bonded to R⁸)

(wherein R⁵ represents hydrogen, C1-3 alkyl or a 5- to 8-memberedheterocyclic skeleton containing 1-3 heteroatoms which is bonded to R⁶or R⁹, R⁶ represents hydrogen, optionally substituted C1-3 alkyl, aC5-10 hydrocarbon ring which is bonded to R⁸ or a 5- to 8-memberedheterocyclic skeleton containing 1-3 heteroatoms which is bonded to R⁵or R⁹, and R⁷ represents hydrogen or optionally substituted C1-6 alkyl);R⁸ represents hydrogen, carboxyl, optionally substituted C1-6 alkyl, a5- to 8-membered heterocyclic skeleton containing 1-3 heteroatoms whichis bonded to R⁴ or a C5-10 hydrocarbon ring which is bonded to R⁶; R⁹represents hydrogen, optionally substituted C1-6 alkyl, optionallysubstituted C1-6 alkyloxycarbonyl, optionally substituted acyl or a 5-to 8-membered heterocyclic skeleton containing 1-3 heteroatoms which isbonded to R⁵ or R⁶; and R¹⁰ represents hydrogen or optionallysubstituted C1-6 alkyl)

In general formula (3) above, C1-3 alkyl may be straight-chain orbranched, and specifically there may be mentioned methyl, ethyl,n-propyl, isopropyl and the like. C1-6 alkyl may be straight-chain orbranched, and specifically there may be mentioned methyl, ethyl,n-propyl, isopropyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, hexyland the like. As C5-10 hydrocarbon rings there may be mentionedcyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl,cyclodecanyl and the like.

As heteroatoms for the 5- to 8-membered heterocyclic skeleton containing1-3 heteroatoms there may be mentioned nitrogen, oxygen, sulfur and thelike, among which nitrogen and oxygen are particularly preferred, and as5- to 8-membered heterocyclic skeletons there may be mentionedpyrrolidine, piperidine, imidazolidine, piperazine, tetrahydrofuran,tetrahydropyran, tetrahydrothiophene, morpholine and the like.

As C1-6 alkyloxycarbonyl there may be mentioned methyloxycarbonyl,ethyloxycarbonyl, isopropyloxycarbonyl, isobutyloxycarbonyl,t-butyloxycarbonyl and the like. As acyl there may be mentioned formyl,acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, valeryl andthe like. When the aforementioned C1-3 or C1-6 alkyl, C1-6alkyloxycarbonyl or acyl have substituents there are no particularrestrictions on the types, positions and numbers of substituents, and asexamples of substituents there may be mentioned halogen such as fluorineand chlorine, hydroxyl, alkyl, carboxyl, amino, alkoxy, nitro, aryl andthe like. As pharmaceutically acceptable salts there may be mentionedsalts of inorganic acids such as hydrochloric acid, sulfuric acid,nitric acid and phosphoric acid, salts of organic acids such as aceticacid and citric acid, salts of inorganic bases such as Na, K, Mg, Ca andammonia, and salts of organic bases such as triethylamine andcyclohexylamine.

As examples of compounds represented by general formula (3) above theremay be mentioned 1-acetylamino-2-hydroxypropane,1-methylamino-2-hydroxypropane, 1-amino-2-oxopropane,1-amino-2-hydroxycyclopentane, 1-amino-2,3-dihydroxypropane,L-threonine, 4-amino-3-hydroxybutanoic acid, 1-amino-2-oxocyclohexane,morpholine, 3-hydroxypyrrolidine, 3-hydroxypiperidine,2-aminomethyl-tetrahydrofuran,1-(2-hydroxypropyl)amino-2-hydroxypropane,1-t-butyloxycarbonylamino-2-hydroxypropane, 2-amino-3-hydroxybutane,DL-serine, 1-amino-2-hydroxypropane, 1-amino-2-hydroxybutane and1-amino-2-hydroxycyclohexane. Compounds among these having asymmetriccarbons may be optically active forms or racemic forms, unless otherwisespecified.

Addition of such activity inducers to the medium can induce cellularactivity and thus more efficiently promote production of the opticallyactive β-aminoalcohol than when no such activity inducers are added. Theactivity inducers may be used alone, or several such activity inducersmay be used in admixture. The amount of such activity inducers ispreferably 0.01-10 wt% with respect to the medium.

The reaction method for production of the β-aminoalcohol of theinvention is not particularly restricted so long as it is a method inwhich the cells or the cell-produced enzyme is reacted with anenantiomeric mixture of an α-aminoketone compound represented by generalformula (1) above or its salt, to produce the corresponding opticallyactive β-aminoalcohol compound represented by general formula (2), andthe reaction is initiated by mixing the cells rinsed with buffersolution or water with the α-aminoketone aqueous solution used as thestarting material.

The reaction conditions may be selected within a range that does notimpede production of the optically active β-aminoalcohol compoundrepresented by general formula (2). The cell volume is preferably 1/100to 1000-fold and more preferably 1/10 to 100-fold in terms of dry weightwith respect to the racemic aminoketone. The concentration of theracemic aminoketone substrate is preferably 0.01-20% and more preferably0.1-10%. The pH of the reaction solution is preferably 5-9 and morepreferably 6-8, and the reaction temperature is preferably 10-50° C. andmore preferably 20-40° C. The reaction time is preferably 5-150 hours,but this may be appropriately determined depending on the cell type.

In order to more efficiently promote the reaction, there may be addedsugars such as glucose, organic acids such as acetic acid and energysources such as glycerol. These may be used alone or as mixtures. Theamount of addition is preferably 1/100 to 10-fold with respect to thesubstrate. Coenzymes and the like may also be added. As coenzymes theremay be used nicotinamide adenine dinucleotide (NAD), reducednicotinamide adenine dinucleotide (NADH), nicotinamide adeninedinucleotide phosphate (NADP), reduced nicotinamide adenine dinucleotidephosphate (NADPH) and the like, either alone or in mixtures, added inamounts of preferably 1/1000 to ⅕ with respect to the racemicaminoketone. In addition to such coenzymes, there may be addedcoenzyme-regenerating enzymes such as glucose dehydrogenase, in amountsof 1/1000 to ⅕ with respect to the racemic aminoketone. Also, substratesfor coenzyme-regenerating enzymes, such as glucose, may be added, inamounts of 1/100 to 10-fold with respect to the racemic aminoketone.There may also be used combinations of sugars such as glucose, organicacids such as acetic acid, energy sources such as glycerol, coenzymes,coenzyme-regenerating enzymes and coenzyme-regenerating enzymesubstrates. These usually accumulate in the cells but if necessary theymay be added to increase the reaction speed or yield, and therefore maybe added as appropriate.

If the reaction solution is reacted with addition of the specific saltsdescribed above under the aforementioned conditions, racemization of theunreacted α-aminoketone isomers will be aided, thus more efficientlypromoting conversion to the enantiomer which will serve as the substrateof the cells or cell-produced enzyme. This will tend to yield the targetaminoalcohol from the starting material at a high yield of 50% orgreater.

As salts that promote racemization of unreacted α-aminoketones there maybe used weak acid salts such as acetate, tartarate, benzoate, citrate,malonate, phosphate, carbonate, paranitrophenol salt, sulfite andborate, but there are preferably used phosphate (for example, sodiumdihydrogen phosphate, potassium dihydrogen phosphate, ammoniumdihydrogen phosphate), carbonate (for example, sodium carbonate, sodiumhydrogen carbonate, potassium carbonate, ammonium carbonate) and citrate(for example, sodium citrate, potassium citrate, ammonium citrate).Mixtures thereof may also be used, with a buffer solution with a pH of6.0-8.0 added to a final concentration of preferably 0.01-1 M. In thecase of a phosphate, for example, sodium dihydrogen phosphate and sodiummonohydrogen phosphate may be mixed in a proportion of between 9:1 and5:95.

The optically active α-aminoalcohol produced by the reaction may bepurified by ordinary separation and purification means. For example, theoptically active β-aminoalcohol may be obtained directly from thereaction solution or after separation of the cells, by being subjectedto a common purification process such as membrane separation, extractionwith an organic solvent (for example, toluene, chloroform, etc.), columnchromatography, vacuum concentration, distillation, crystallization,recrystallization or the like. The optical purity of the producedoptically active β-aminoalcohol can be measured by high performanceliquid chromatography (HPLC).

EXAMPLES

The present invention will now be explained in greater detail throughexamples, with the understanding that these examples in no way limit thetechnical scope of the invention.

Example 1 Isolation and Purification of Plasmids

(1) Method

Rhodococcus strains were inoculated to 5 mL of GPY medium (1% glucose,0.5% bactopeptone, 0.3% yeast extract) and cultured with shaking at 25°C. After adding 250 μL of a 100 mg/mL ampicillin solution in thelogarithmic growth phase, culturing was continued at 25° C. for 2 hourswith shaking. The cells were harvested by centrifugation (12 krpm, 5min), and after removing off the supernatant, they were suspended in 1mL of 50 mM Tris (pH 7.5), the cells were again harvested bycentrifugation (12 krpm, 5 min) and the supernatant was removed off.They were then suspended in 250 μL of a 10 mg/mL lysozyme solutiondissolved in TE solution (10 mM Tris (pH 7.5), 1 mM EDTA), and thesuspension was allowed to stand at 37° C. for 30 minutes. Next, 100 μLof 3 M sodium chloride and 25 μL of 10% SDS were added and the mixturewas allowed to stand at −20° C. overnight. To the supernatant fromcentrifugation (12 krpm, 5 min) there were added 0.5 μL each of 50 μg/mLProteinase K and 50 μg/mL RNase A, and the mixture was allowed to standat 37° C. for 15 minutes. An equivalent of phenol/chloroform/isoamylalcohol solution was added and centrifugation was performed (12 krpm, 5min). A 2.5-fold amount of ethanol was added to the supernatant, themixture was centrifuged (12 krpm, 5 min), and the precipitate wasdissolved in 50 μL of sterilized water. Confirmation of plasmids wasaccomplished by electrophoresis with 0.8% agarose gel and staining withethidium bromide, followed by UV irradiation.

(2) Test Bacteria Strains and Results

Throughout the examples, the presence or absence of plasmids wasscreened from available strains belonging to the genus Rhodococcus andits related genus Mycobacterium followed the method described in (1)above.

Table 3 shows the screened strains confirmed to contain plasmids.Specifically, Rhodococcus erythropolis (IAM1400, IAM1503, JCM2893,JCM2894 and JCM2895) and Rhodococcus rhodnii (JCM3203) were confirmed tocontain plasmids of approximately 5.4 kbp and 5.8 kbp, respectively.These plasmids were designated according to the names listed in Table 3:pRET1100, pRET1200, pRET1300, pRET1400, pRET1500, pRET1600, pRET1700,pRET1800, pRET0500, pRET1000 (see Table 3).

R. erythropolis IAM1400 and IAM1503 are described in “IAM Catalogue ofStrains, Third Edition, 2004” published by the Institute of Molecularand Cellular Biosciences, The University of Tokyo, and are availablefrom the institute. Also, R. erythropolis JCM2893, JCM2894 and JCM2895and R. rhodnii JCM3203 are described in “JCM Catalogue of Strains,Eighth Edition 2002” published by RIKEN, Japan, and are available fromthe institute.

TABLE 3 Strain No. Size (kbp) Name Rhodococcus erythropolis IAM 1400 5.4pRET1100 5.4 pRET1200 ″ IAM 1503 5.4 pRET1300 5.4 pRET1400 ″ JCM 28935.4 pRET1500 5.4 pRET1600 ″ JCM 2894 5.4 pRET1700 5.4 pRET1800 ″ JCM2895 5.4 pRET0500 Rhodococcus rhodnii JCM 3203 5.8 pRET1000

Example 2 Identification of Restriction Endonuclease Sites

Various restriction endonucleases were used to determine restrictionendonuclease sites, for classification of the plasmids shown in Table 3.Each plasmid was isolated by the method described in Example 1, and thendigested with EcoR I, Hind III, Pvu II, Sca I, Sph I, Sma I, Sac I, BamHI and Kpn I, and electrophoresed on 0.8% agarose gel for confirmation ofthe DNA fragments. The size marker used was Loading Quick DNA sizeMarker λ/EcoR I+Hind III double digest (Toyobo). The numbers of sitescleaved by the restriction endonucleases and the sizes of the fragmentswere determined based on the size marker. The results are shown in Table4.

TABLE 4 R. erythropolis JCM R. rhodnii IAM 1400 IAM 1503 JCM 2893 JCM2894 2895 JCM 3203 pRET1100 pRET1200 pRET1300 pRET1400 pRET1500 pRET1600pRET1700 pRET1800 pRET0500 pRET1000 BamH I 2(0.4, 5.0) 1(5.4) same samesame same same same same 2(2.0, 3.8) EcoR I 2(0.3, 5.1) 1(5.4) as as asas as as as 0 Hind III 0 0 pRET1100 pRET1200 pRET1100 pRET1200 pRET1100pRET1200 pRET1200 0 Kpn I 1(5.4) 0 0 Pvu II 1(5.4) 2(0.9, 4.5) 4(0.1,1.4, 2.0, 2.3) Sac I 1(5.4) 1(5.4) 3(0.9, 1.0, 3.9) Sca I 0 0 0 Sph I 00 0 Sma I 1(5.4) 2(0.4, 0.5) 4(0.1, 1.2, 1.6, 2.9) Values in parenthesesindicate sizes (kbp)

Based on the analysis results shown above, the plasmids in Table 3 wereclassified into three types: plasmids possessing the same restrictionendonuclease sites as pRET1100, plasmids possessing the same restrictionendonuclease sites as pRET1200, and pRET1000.

Example 3 Plasmid Sequencing and Homology Search

As the plasmids were classified into three types, i.e. pRET1000,pRET1100 and pRET1200 based on the results of Example 2, it wasattempted to sequence each of the plasmids.

First, the DNA fragments of the plasmids were cloned for determinationof the nucleotide sequences. For Rhodococcus erythropolis (IAM1400), theplasmids (pRET1100, pRET1200) were isolated and digested with Sma I andSac I. Upon electrophoresis on 0.8% agarose gel, DNA fragments withsizes of approximately 0.5 kbp, approximately 1.7 kbp, approximately 3.7kbp and approximately 4.9 kbp were confirmed. The respective DNAfragments were recovered from the agarose gel using a GFX™ PCR DNA andGel Band Purification Kit (Amersham Bioscience) and used as insert DNA.Separately, pBluescript II KS(−) was used after digesting with Sma Ialone or with Sma I and Sac I, as vector DNA. The insert DNA and vectorDNA were ligated with Ligation High (Toyobo) and used to transform E.coli JM109. The obtained transformants were screened using a GFX MicroPlasmid Prep Kit (Amersham Bioscience) to obtain different clones.

For Rhodococcus rhodnii (JCM3203), the plasmid (pRET1000) was isolatedand then digested with BamH I. Upon electrophoresis on 0.8% agarose gel,DNA fragments with sizes of approximately 2.0 kbp and approximately 3.8kbp were confirmed. The respective DNA fragments were recovered from thegel using the aforementioned Kit and used as insert DNA. The vector DNAused was pBluescript II KS(−) digested with BamH I.

Determination of the nucleotide sequences of the plasmid inserts wasaccomplished by the primer walking method. The apparatus used was an ABIPRISM™310NT Genetic Analyzer, and the enzyme used was a BigDyeTerminator v3.1 Cycle Sequencing Kit (ABI).

First, P7 (M13 forward, Toyobo) and P8 (M13 reverse, Toyobo) primerswere used for partial decoding of the insert nucleotide sequences. Next,primers were designed within the decoded sequence (using the sequenceanalyzing software DNASIS Pro; Hitachi Software Corp.), and the designedprimers (synthetic oligo DNA) were used for further decoding of thenucleotide sequence. This procedure was repeated until decoding of theentirety of each insert nucleotide sequence. Upon completion of theinsert nucleotide sequence decoding, primers were designed for reactionfrom the ends of each insert to the vector direction in order to analyzehow the inserts were linked, and PCR was conducted (using KOD -plus-),using the plasmid isolated from Rhodococcus erythropolis (IAM1400) astemplate. The PCR product was purified using a GFX™ PCR DNA and Gel BandPurification Kit, and sequencing was carried out using the same primersused for PCR, to analyze the arrangement of the inserts.

The results of sequencing showed that pRET1100 consisted of 5444 bp,with a G+C content of 59%. The full determined nucleotide sequence isset forth as SEQ ID NO: 73 of the Sequence Listing. Plasmid pRET1200consisted of 5421 bp and had a G+C content of 62%. Plasmid pRET1000consisted of 5813 bp and had a G+C content of 67%. The full determinednucleotide sequence is set forth as SEQ ID NO: 74 of the SequenceListing.

A homology search for the determined nucleotide sequences using DNASISPro revealed that pRET1000 and pRET1100 were novel plasmids. On theother hand, pRET1200 had approximately 99.6% homology with pN30 (GenBankaccession no. AF312210) (calculated based on pRET1200).

For pRET1000 and pRET1100, comparison was made with publicly knownplasmids based on the determined nucleotide sequences, using DNASIS Pro.As a result, neither of the plasmids were found to have completelymatching restriction endonuclease sites with other plasmids.

Example 4 Nucleotide Sequence Analysis

The results of analysis of the nucleotide sequences of pRET 1100 andpRET1000 are shown below.

The following orfs were found in pRET1100:

-   -   orf1 (SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) consisting of        the nucleotide sequence from bases 202, 238 or 337 to 480 of the        nucleotide sequence set forth as SEQ ID NO: 73;,    -   orf2 (SEQ ID NO: 4) consisting of the nucleotide sequence from        bases 477 to 758 of the nucleotide sequence set forth as SEQ ID        NO: 73;    -   orf3 (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,        SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ        ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16)        consisting of the nucleotide sequence from bases 862, 1294,        1450, 1462, 1486, 1489, 1513, 1630, 1645, 1687, 2224 or 2227 to        2409 of the nucleotide sequence set forth as SEQ ID NO: 73;    -   orf4 (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20        or SEQ ID NO: 21) consisting of the nucleotide sequence        complementary to the nucleotide sequence from bases 1875, 1734,        1701, 1674 or 1581 to 1444 of the nucleotide sequence set forth        as SEQ ID NO: 73;    -   orf5 (SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25        or SEQ ID NO: 26) consisting of the nucleotide sequence        complementary to the nucleotide sequence from bases 2828, 2792,        2747, 2594 or 2540 to 2406 of the nucleotide sequence set forth        as SEQ ID NO: 73;    -   orf6 (SEQ ID NO: 27 or SEQ ID NO: 28) consisting of the        nucleotide sequence from bases 2971 or 3049 to 3306 of the        nucleotide sequence set forth as SEQ ID NO: 73;    -   orf7 (SEQ ID NO: 29 or SEQ ID NO: 30) consisting of the        nucleotide sequence complementary to the nucleotide sequence        from bases 3577 or 3571 to 3053 of the nucleotide sequence set        forth as SEQ ID NO: 73;    -   orf8 (SEQ ID NO: 31 or SEQ ID NO: 32) consisting of the        nucleotide sequence from bases 3339 or 3648 to 3902 of the        nucleotide sequence set forth as SEQ ID NO: 73; and    -   orf9 (SEQ ID NO: 33 or SEQ ID NO: 34) consisting of the        nucleotide sequence from bases 4366 or 4477 to 5034 of the        nucleotide sequence set forth as SEQ ID NO: 73.

The following orfs were found in pRET1000:

-   -   orf10 (SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID        NO: 41) consisting of the nucleotide sequence complementary to        the nucleotide sequence from bases 3350, 3251, 2945 or 2849 to        2412 of the nucleotide sequence set forth as SEQ ID NO: 74;    -   orf11 (SEQ ID NO: 42 or SEQ ID NO: 43) consisting of the        nucleotide sequence complementary to the nucleotide sequence        from bases 2365 or 2332 to 2159 of the nucleotide sequence set        forth as SEQ ID NO: 74;    -   orf12 (SEQ ID NO: 44) consisting of the nucleotide sequence from        bases 3197 to 3526 of the nucleotide sequence set forth as SEQ        ID NO: 74;    -   orf13 (SEQ ID NO: 45 or SEQ ID NO: 46) consisting of the        nucleotide sequence complementary to the nucleotide sequence        from bases 4035 or 3996 to 3679 of the nucleotide sequence set        forth as SEQ ID NO: 74;    -   orf14 (SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50) consisting        of the nucleotide sequence from bases 4621, 4654 or 4666 to 4830        of the nucleotide sequence set forth as SEQ ID NO: 74;    -   orf15 (SEQ ID NO: 51 or SEQ ID NO: 52) consisting of the        nucleotide sequence complementary to the nucleotide sequence        from bases 5161 or 5062 to 4709 of the nucleotide sequence set        forth as SEQ ID NO: 74;    -   orf16 (SEQ ID NO: 53 or SEQ ID NO: 54) consisting of the        nucleotide sequence from bases 2331 or 2334 to 2618 of the        nucleotide sequence set forth as SEQ ID NO: 74;    -   orf17 (SEQ ID NO: 55) consisting of the nucleotide sequence from        bases 2907 to 3242 of the nucleotide sequence set forth as SEQ        ID NO: 74;    -   orf18 (SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:        59 or SEQ ID NO: 60) consisting of the nucleotide sequence from        bases 1650, 1689, 1713, 1827 or 1875 to 2162 of the nucleotide        sequence set forth as SEQ ID NO: 74;    -   orf19 (SEQ ID NO: 61) consisting of the nucleotide sequence from        bases 1906 to 2169 of the nucleotide sequence set forth as SEQ        ID NO: 74;    -   orf20 (SEQ ID NO: 62) consisting of the nucleotide sequence        complementary to the nucleotide sequence from bases 810 to 553        of the nucleotide sequence set forth as SEQ ID NO: 74;    -   orf21 (SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:        66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69) consisting of        the nucleotide sequence from bases 117, 147, 306, 456, 5144,        5276 or 5534 to 656 of the nucleotide sequence set forth as SEQ        ID NO: 74.

The DNA replication region of pRET1100 is the region represented by thenucleotide sequence set forth as SEQ ID NO: 35 (from bases 2410 to3200), the nucleotide sequence set forth as SEQ ID NO: 36 (from bases1000 to 1500) or the nucleotide sequence set forth as SEQ ID NO: 37(from bases 5000 to 500). The DNA replication region of pRET1000 is theregion represented by the nucleotide sequence set forth as SEQ ID NO: 70(from bases 3355 to 3507), the nucleotide sequence set forth as SEQ IDNO: 71 (from bases 4290 to 4350) or the nucleotide sequence set forth asSEQ ID NO: 72 (from bases 3570 to 3894).

The region of the nucleotide sequence from bases 5144 to 656 (SEQ ID NO:67) and the region of the nucleotide sequence from bases 4381 to 4830(SEQ ID NO: 47) of the nucleotide sequence of pRET1000 (SEQ ID NO: 74)are homologous with mobilization proteins, suggesting that they areinvolved in mobilization.

A DNA secondary structure is predicted for the region of the nucleotidesequence from bases 4260 to 4339 (SEQ ID NO: 75) of the nucleotidesequence of pRET1000 (SEQ ID NO: 74), and it is presumably involved inexpression of the mobilization protein gene or is the recognition siteof the expressed protein.

On the other hand, it was suggested that the region of the nucleotidesequence from bases 761 to 868 (SEQ ID NO: 76) of the nucleotidesequence of pRET1100 (SEQ ID NO: 73) is a promoter involved inexpression of a protein related to replication.

Example 5 Construction of Shuttle Vectors

For construction of a shuttle vector between Rhodococcus strains and E.coli, the Rhodococcus plasmids pRET1000, pRET1100 and pRET1200 and theE. coli plasmids pUC18, pHSG299 and pHSG398 were used for the followingexperiment.

First, DNA fragments were prepared from R. erythropolis plasmids.Specifically, plasmids pRET1100 and pRET1200 were obtained from R.erythropolis (IAM1400), and then Alw44 I was used for digestion ofpRET1100 at 37° C. for 2 hours and Blunting High (Toyobo) was used forblunting of the ends, while BspLU11 I was used for digestion of pRET1200at 48° C. for 2 hours and Blunting High (Toyobo) was used for bluntingof the ends, to obtain DNA fragments of R. erythropolis plasmid. Each ofthe DNA fragments was dissolved in TE solution.

For pRET1000, plasmid pRET1000 was obtained from R. rhodnii (JCM3203),and then Drd I was used for digestion of pRET1000 at 37° C. for 2 hoursand Blunting High was used for blunting of the ends, to obtain pRET1000DNA fragments, which were dissolved in TE solution.

Next, DNA fragments were prepared from the E. coli plasmids.Specifically, pUC18 (containing the ampicillin-resistance gene(Amp^(r))) was digested with Sma I at 30° C. for 2 hours, and pHSG299(containing the kanamycin-resistance gene (Km^(r))) and pHSG398(containing the chloramphenicol-resistance gene (Cm^(r))) were digestedwith Hinc II at 37° C. for 2 hours to obtain DNA fragments of E. coliplasmid, which were dissolved in TE.

After ligating the DNA fragments from the Rhodococcus and E. coliplasmids prepared in the manner described above, they were used fortransformation in E. coli DH5α, which were plated on LB (1% tryptophan,0.5% yeast extract, 1% sodium chloride; pH 7.2) agar medium containing100 μg/mL kanamycin, 100 μg/mL ampicillin or 30 μg/mL chloramphenicol,coated with 30 μL of 0.1 M IPTG (isopropyl-β-galactoside) and 4% X-gal(5-bromo-4-chloro-3-indole-β-D-galactopyranoside) and allowed to standat 30° C. for 60 hours. White colonies were selected from among theappearing colonies, and were cultured with shaking in LB liquid mediumcontaining 100 μg/mL kanamycin, 100 μg/mL ampicillin or 30 μg/mLchloramphenicol, at 30° C. for 60 hours. The DNA was purified from theobtained culture solution using a GFX™ Micro Plasmid Prep Kit (AmershamBioscience, with purification under the manufacturer's specifiedconditions). The obtained DNA was confirmed by electrophoresis on 0.8%agarose gel. The obtained shuttle vectors are shown in Table 5, and themethods for constructing each of the shuttle vectors using pRET1100 areshown in FIGS. 3 to 5.

TABLE 5 Constructed shuttle Origin vectors Rhodococcus E. coli pRET1001,pRET1001Rv pRET1000 pUC18 pRET1002, pRET1002Rv pRET1000 pHSG299pRET1003, pRET1003Rv pRET1000 pHSG398 pRET1101, pRET1101Rv pRET1100pUC18 pRET1102, pRET1102Rv pRET1100 pHSG299 pRET1103, pRET1103RvpRET1100 pHSG398 pRET1201, pRET1201Rv pRET1200 pUC18 pRET1202,pRET1202Rv pRET1200 pHSG299 pRET1203, pRET1203Rv pRET1200 pHSG398

The shuttle vectors constructed with pRET1100 and pUC18, pHSG299 orpHSG398 were designated respectively as pRET1101 (SEQ ID NO: 89),pRET1102 (SEQ ID NO: 90) or pRET1103 (SEQ ID NO: 91), respectively. Ofthe shuttle vectors, pRET1101 exhibits ampicillin resistance, pRET1102exhibits kanamycin resistance and pRET1103 exhibits chloramphenicolresistance. Also, the shuttle vectors pRETI 101 to 1103 wherein the E.coli gene and pRET1100 were linked in reverse (Rv) were designatedrespectively as pRET1101Rv (SEQ ID NO: 92), pRET1102Rv (SEQ ID NO: 93)and pRET1103Rv (SEQ ID NO: 94).

Similarly, the shuttle vectors constructed using pRET1000 and pRET1200were designated as pRET1001-pRET1003 (SEQ ID NO: 95-SEQ ID NO: 97) andpRET1001Rv-pRET1003Rv (SEQ ID NO: 98-SEQ ID NO: 100), and aspRET1201-pRET1203 and pRET1201Rv-pRET1203Rv (Table 5).

Example 6 Examining Method of Transformation to R. erythropolis

The Rhodococcus-E. coli shuttle vectors obtained in Example 5 were usedfor transformation of R. erythropolis MAK-34 strain (MAK-34; depositedat the National Institute of Bioscience and Human-Technology, NationalInstitute of Advanced Industrial Science and Technology, Ministry ofEconomy, Trade and Industry, (currently: International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology) on Feb. 15, 2001 as FERM BP-7451). Electroporation wasinvestigated as the method of gene transfer.

First, R. erythropolis MAK-34 strain was inoculated to 5 mL of GPYmedium and cultured with shaking at 30° C. for 36 hours. After seeding 1mL of culture solution in 100 mL of LB medium, culturing was continuedat 200 rpm at 30° C. for 10 hours. The cultured cells were harvested bycentrifugation (12 krpm, 5 min, 4° C.) and the harvested cells wererinsed twice with ultrapurified water. The rinsed cells were harvestedby centrifugation (12 krpm, 5 min, 4° C.) and suspended in 2.4 mL of a10% glycerol solution. The suspension was dispensed into 300 μl portionsand frozen at −80° C. as competent cells.

A 90 μL portion of the prepared competent cells and a 5 μL portion ofthe shuttle vector (pRET1001, pRET1002, pRET1003, pRET1101, pRET1102,pRET1103, pRET1201, pRET1202 or pRET1203) were mixed on ice. The mixedsolution was gently poured into a 0.1 cm cuvette which had been cooledon ice, and was set in a Gene Pulser II Electroporation System(BIO-RAD). After pulsing at 20 kV/cm, 400Ω, 25 μF, the mixed solutionwas added with 300 μL of LB medium immediately and was allowed to standat 25° C. for 3 hours.

A portion of the cell suspension was plated on an antibiotic-containingLB plate (100 μg/mL kanamycin, 100 μg/mL ampicillin or 30 μg/mLchloramphenicol). As a result, colonies were obtained when usingpRET1002, pRET1102 and pRET1202 containing the kanamycin resistancegene. In order to confirm that the obtained colonies contained theplasmids, the plasmids were isolated and all were verified to containthe shuttle vector.

This suggested that R. erythropolis can be transformed byelectroporation and that pRET1002, pRET1102 and pRET1202 function asshuttle vectors.

Example 7 Obtaining Aminoketone Asymmetric Reductase Gene (Mak Gene)

The mak gene was isolated from R. erythropolis MAK-34 strain forinsertion of the mak gene into the shuttle vector shown in FIG. 5.

First, genomic DNA was obtained from R. erythropolis MAK-34 strain.After inoculating R. erythropolis MAK-34 strain to 5 mL of GPY medium,culturing with shaking was performed at 30° C. for 48 hours, and thenthe culture solution was seeded in 100 mL of GPY medium and subculturedat 200 rpm at 30° C. for 10 hours. The genomic DNA was obtained using aGenomic DNA Buffer set and Genomic-tip 500/G (QIAGEN).

The obtained genomic DNA was used as template for PCR using KOD -plus-.The primers used were MAKF1 (5′-GAATCTTCTCGTTGATGCAGATCAGGTC-3′; SEQ IDNO: 80) and MAKR² (5′-CTGACTCCGTAGTGTTCTGCCAGTTC-3′; SEQ ID NO: 81), forPCR at an annealing temperature of 68° C. and extension reaction for 1minute and 50 seconds. The obtained PCR product was subjected tophenol/chloroform treatment and ethanol precipitation, and then mixedwith pUC18 that had been digested with Sma I for 2 hours at 30° C., andligated therewith using Ligation High. Competent High (Toyobo) was usedfor transformation of E. coli DH5α, which was then plated on LB agarmedium (containing 100 μg/mL ampicillin) that had been coated with 30 μLof 0.1 M IPTG and 4% X-gal, and was allowed to stand at 30° C. for 60hours. White colonies were selected from among the appearing colonies,and were cultured with shaking in LB liquid medium containing 100 μg/mLampicillin at 30° C. for 60 hours. The DNA was purified from theobtained culture solution using a GFX™ Micro Plasmid Prep Kit. Theobtained DNA was confirmed by electrophoresis on 0.8% agarose gel. Theobtained clone was designated as pMAK-1.

Example 8 Construction of Expression Vector-1

A promoter and aminoketone asymmetric reductase gene (mak gene) wereinserted into the shuttle vector shown in Table 5.

First, an expression vector (without exogenous promoter) containingapproximately 400 bp upstream from the mak gene was constructed.

pMAK-1 was digested with Sma I at 30° C. for 2 hours, and then with PstI at 37° C. for 2 hours. The solution was supplied for 0.8% agarose gelelectrophoresis. The DNA size marker used was Loading Quick DNA sizeMarker λ/EcoR I+Hind III double digest. After electrophoresis, anapproximately 1.4 kbp DNA fragment was purified using a GFX™ PCR DNA andGel Band Purification Kit, and used as the insert DNA. On the otherhand, the vector used was pRET1102 digested with Hinc II and Pst I at37° C. for 2 hours. The DNA fragments were ligated with Ligation Highand Competent High was used for transformation of E. coli DH5α. Thecells were plated on LB agar medium containing 100 μg/mL kanamycin andallowed to stand at 30° C. for 60 hours.

The appearing colonies were cultured with shaking on LB liquid mediumcontaining 100 μg/mL kanamycin at 30° C. for 60 hours. The DNA waspurified from the obtained cultured medium using a GFX™ Micro PlasmidPrep Kit. The obtained DNA was confirmed by 0.8% agarose gelelectrophoresis.

For screening, the obtained DNA without restriction endonucleasetreatment and the DNA after digestion with Pst I at 37° C. for 2 hourswere subjected to 0.8% agarose gel electrophoresis, and the targetplasmid was obtained based on the size of the DNA. The size marker usedwas Loading Quick DNA size Marker λ/EcoR I+Hind III double digest,pRET1102 and pRET1102 that had been digested with Pst I at 37° C. for 2hours. The plasmid obtained in this manner was designated as pRET1104.

Example 9 Construction of Expression Vector-2

The shuttle vectors were reduced, since reduction of shuttle vectors iseffective for expression vector enhancement, gene modification,transformation efficiency improvement and replication in cells.

First, shuttle vector pRET1102 was reduced. After digesting pRET1102with BamH I and Hinc II for 2 hours, it was electrophoresed on 0.8%agarose gel and an approximately 2.7 kbp DNA fragment was recoveredusing a GFX™ PCR DNA and Gel Band Purification Kit to prepare a pRET1102DNA fragment. The size marker used was Loading Quick DNA size Markerλ/EcoR I+Hind III double digest.

Separately, a DNA fragment replicable in E. coli was prepared bydigesting pHSG299 with BamH I and Hinc II for 2 hours, subjecting it to0.8% agarose gel electrophoresis, and recovering an approximately 2.7kbp DNA fragment using a GFX™ PCR DNA and Gel Band Purification Kit.

The DNA fragments were ligated with Ligation High and Competent High wasused for transformation of E. coli JM109 cells, which were then platedon LB agar medium, containing 100 μg/mL kanamycin, that had been coatedwith 30 μL of 0.1 M IPTG and 4% X-gal, and was allowed to stand at 30°C. for 48 hours.

White colonies were selected from among the appearing colonies, and werecultured with shaking in LB liquid medium containing 100 μg/mL kanamycinat 30° C. for 48 hours. The DNA was purified from the obtained culturesolution using a GFX™ Micro Plasmid Prep Kit. The reduced shuttle vectorof pRET1102 obtained in this manner was designated as pRET1123(approximately 5.3 kbp).

Next, shuttle vector pRET1202 was reduced. The Rhodococcus-derived DNAfragment was prepared by digesting pRET1202 with EcoR I for 2 hours andthen with Dra III for 2 hours, using Blunting High for blunting of theends, performing 0.8% agarose gel electrophoresis, and then recoveringan approximately 3.7 kbp DNA fragment using a GFX™ PCR DNA and Gel BandPurification Kit. The size marker used was Loading Quick DNA size Markerλ/EcoR I+Hind III double digest. The DNA fragment was inserted at theHinc II site of pHSG299. After ligation, Competent High was used fortransformation of E. coli DH5α, which was then plated on LB agar medium,containing 100 μg/mL kanamycin, that had been coated with 30 μL of 0.1 MIPTG and 4% X-gal, and was allowed to stand at 30° C. for 72 hours.White colonies were selected from among the appearing colonies, and werecultured with shaking in LB liquid medium containing 100 μg/mL kanamycinat 30° C. for 72 hours. The DNA was purified from the obtained culturesolution using a GFX™ Micro Plasmid Prep Kit. When the plasmid obtainedby screening was digested with Sac I, BamH I, Pst I or EcoR I for 2hours, all of the clones had approximately 500 bp clipped at the side ofEcoR I site of the Rhodococcus-derived region. The plasmid wasdesignated as pRET1204 (approximately 5.9 kbp). It was not possible toobtain a clone with no clipping of the genus Rhodococcus replicationregion.

The shuttle vector pRET1002 was reduced in a similar manner to obtainpRET1006 (approximately 4.9 kbp).

R. erythropolis was transformed with these three reduced plasmids,pRET1006, pRET1123 and pRET1204, and upon confirming the presence orabsence of shuttle vector by the method described in Example 6, all theshuttle vectors were detected in the transformed cells. This suggestedthat the three reduced plasmids pRET1006, pRET1123 and pRET1204 arereplicated in R. erythropolis.

Example 10 Construction of Expression Vector-3

An expression vector was constructed by having the mak gene insertedinto the shuttle vector constructed in Example 9.

The Pst I site of pRET1123 constructed in Example 9 was deleted forcloning of the promoter in the single step. After digesting pRET1123with Pst I for 2 hours, Blunting High was used for blunting of the endsand Ligation High was used for ligation. The solution was used totransform E. coli JM109 using Competent High, and culturing wasperformed on an LB plate containing 100 μg/mL kanamycin at 30° C. for 36hours. The formed colonies were inoculated on LB liquid mediumcontaining 100 μg/mL kanamycin and cultured at 30° C. for 24 hours, andthen the DNA was purified using a GFX™ Micro Plasmid Prep Kit to obtainpRET1132

The obtained pRET1132 was digested with Pst I for 1 hour and thenelectrophoresed on 0.8% agarose, which resulted in confirming lack ofcleavage of pRET1132 by Pst I. As controls there were used pRET1123 andpRET1132 not digested with Pst I, and pRET1123 digested with Pst I.

Example 11 Construction of Expression Vector-4

A clone was constructed having a promoter and the mak gene inserted inthe aforementioned shuttle vector.

A clone was constructed having a Pst I site upstream from the mak gene,for insertion of a promoter. The procedure was carried out in thefollowing manner to obtain a clone having His-Tag added to theC-terminus of the aminoketone asymmetric reductase. PCR was conductedwith KOD -plus- using the pMAK-1 obtained in Example 7 as template,MAKPstF (5′-GACCACTGCAGATCAATCAACTCTGATGAGGTCC-3′; SEQ ID NO: 82) andMAKHisBglIIR (5′-CGCTTAGATCTCAGTTCGCCGAGCGCCATCGCCG-3′; SEQ ID NO: 83)as primers, with an annealing temperature of 68° C. and extensionreaction for 1 minute and 50 seconds. A PCR fragment (insert) producedby digesting the obtained PCR product with Bgl II at 37° C. for 2 hourswas ligated with pQE70 (digested with Sph I at 37° C. for 2 hours,blunted with Blunting High and digested with Bgl II at 37° C. for 2hours) using Ligation High, and then Competent High was used fortransformation of E. coli DH5αcells, which were plated on LB agar mediumcontaining 100 μg/mL ampicillin and allowed to stand at 30° C. for 60hours. The appearing colonies were cultured with shaking on LB liquidmedium containing 100 μg/mL ampicillin at 30° C. for 60 hours. The DNAwas purified using a GFX™ Micro Plasmid Prep Kit. The obtained DNA wasconfirmed by 0.8% agarose gel electrophoresis.

For screening, the DNA without restriction endonuclease treatment andthe DNA after digestion with Pst I and Bgl II at 37° C. for 2 hours weresubjected to 0.8% agarose gel electrophoresis, and the target plasmidwas obtained based on the size of the DNA. The plasmid obtained in thismanner was designated as pMAK-2. The size marker used was Loading QuickDNA size Marker λ/EcoR I+Hind III double digest, pQE70, and pQE70 thathad been digested with Bgl II at 37° C. for 2 hours.

A clone was constructed by inserting the pRET1200 repA promoter(obtained by PCR amplification using as template a clone of pRET1204wherein the orientation of repA encoded by the Rhodococcus-derived DNAfragment was in the same orientation as the kanamycin resistance geneencoded by pHSG299, and using as primers P1200rep-Pst5195(5′-AGCCGCTGCAGAAGCAACACCGCATCCGCCCATTG-3′; SEQ ID NO: 84) and P7(5′-CGCCAGGGTTTTCCCAGTCACGAC-3′; SEQ ID NO: 85), with an annealingtemperature of 60° C. and extension reaction for 1 minute, followed bydigestion with EcoR I and Pst I at 37° C. for 2 hours) at the EcoR I-PstI site of pMAK-2 (designated as pMAK-19).

Next, PCR was conducted with KOD -plus- using as template pMAK-19 and asprimers pQE7OF1 (5′-GGCGTATCACGAGGCCCTTTCGTCTTCACC-3′; SEQ ID NO: 86)and pQE70R1135Bm (5′-GGTTGGATCCGTCATCACCGAAACGCGCGAGGCAG-3′; SEQ ID NO:87), with an annealing temperature of 60° C. and extension reaction for3 minutes. The PCR product was purified from the reaction solution byusing a GFX™ PCR DNA and Gel Band Purification Kit and after digestionof the purified PCR product with EcoR I and BamH I for 2 hours, it waselectrophoresed on 0.8% agarose gel and the DNA fragment was purified byusing a GFX™ PCR DNA and Gel Band Purification Kit. The DNA fragment wasused as an insert DNA.

Separately, a vector to be used as the expression shuttle vector wasobtained by digesting pRET1132 with EcoR I and BamH I for 2 hours,subjecting the DNA fragment to 0.8% agarose gel electrophoresis andpurifying the DNA fragment by using a GFX™ PCR DNA and Gel BandPurification Kit. After mixing the insert DNA and vector, Ligation Highwas used for ligating them and Competent High was used fortransformation of E. coli JM109 cells, which were plated on an LB platecontaining 100 μg/mL kanamycin. The obtained colonies were cultured onLB liquid medium containing 100 μg/mL kanamycin, and then the plasmidDNA was recovered by using a GFX™ Micro Plasmid Prep Kit and subjectedto 0.8% agarose gel electrophoresis for screening. The size markers usedwere Loading Quick DNA size Marker λ/EcoR I+Hind III double digest andpRET1132. The obtained expression vector was designated as pRET1133.

Also, pMAK-19 was digested with EcoR I and Hind III at 37° C. for 2hours, blunted with Blunting High and subjected to 0.8% agarose gelelectrophoresis, and the approximately 1.6 kbp DNA fragment was purifiedby using a GFX™ PCR DNA and Gel Band Purification Kit. The clone havingthis fragment inserted at the Hinc II site of pRET1102 was designated aspRET1114.

The pRET1133 promoter was also modified. The mak gene-expressingpromoter encoded in pRET1133 is the repA gene promoter of pRET1200 andhas a length of approximately 800 bp, and a plasmid was constructed byhaving approximately 200 bp clipped off from this promoter. The promoterused for the cloning was prepared by PCR. Plasmid pRET1200 was used astemplate, P1204rep-Ec2958 (5′-CGCGGAATTCGACCACCACGCACGCACACCGCAC-3′; SEQID NO: 88) and P1200rep-Pst5195(5′-AGCCGCTGCAGAAGCAACACCGCATCCGCCCATTG-3′; SEQ ID NO: 84) were used asprimers, and KOD -plus- was used as the PCR enzyme for PCR at anannealing temperature of 60° C. and extension reaction for 50 seconds.The PCR product was purified by using a GFX™ PCR DNA and Gel BandPurification Kit, digested with the restriction endonucleases EcoR I andPst I for 2 hours, and subjected to 1.6% agarose gel electrophoresis,and the DNA fragment Was purified by using a GFX™ PCR DNA and Gel BandPurification Kit. The DNA fragment was used as the insert DNA. Thenucleotide sequence of the promoter region in the DNA fragment is setforth as SEQ ID NO: 77.

Separately, for the vector, pRET1133 was digested with restrictionendonucleases EcoR I and Pst I for 2 hours and subjected to 0.8% agarosegel electrophoresis, and an approximately 7.2 kbp DNA fragment waspurified by using a GFX™ PCR DNA and Gel Band Purification Kit. The sizemarker used was Loading Quick DNA size Marker λ/EcoR I+Hind III doubledigest.

The insert DNA and vector obtained in this manner were ligated by usingLigation High, and Competent High was used for transformation of E. coliJM109 cells, which were plated on an LB plate containing 100 μg/mLkanamycin. The obtained colonies were cultured on LB liquid mediumcontaining 100 μg/mL kanamycin, and then the plasmid DNA was recoveredby using a GFX™ Micro Plasmid Prep Kit and subjected to 0.8% agarose gelelectrophoresis for screening. The size markers used were Loading QuickDNA size Marker λ/EcoR I+Hind III double digest and pRET1133.

Also, after digesting the obtained DNA with restriction endonucleasesEcoR I and Pst I for 2 hours, it was subjected to 1.6% agarose gelelectrophoresis and a DNA fragment corresponding to the approximately600 bp insert DNA was confirmed. The size marker used was a 100 bp DNALadder. The expression vector obtained in this manner was designated aspRET1138.

Example 12 Preparation of Recombinant R. erythropolis and Measurement ofEnzyme Activity

The aforementioned expression vectors pRET1102, pRET1104, pRET1114 andpRET1138 were used for transformation of R. erythropolis MAK-34 strainand R. erythropolis JCM2895 (provided by RIKEN Japan), and the enzymeactivity was measured. The aminoketone asymmetric reductase purifiedfrom MAK-34 strain has the abilities to react with1-2-methylamino-1-phenyl-1-propanone as described in InternationalPatent Publication WO02/070714, and to produce d-(1S,2S)-pseudoephedrine. It was also reacted with1-2-dimethylaminopropiophenone, 1-amino-2-butanone, etc. and productionof each corresponding β-aminoalcohol was confirmed.

The activity assay was conducted by preparing a reaction solution with acell density O.D.=5, 2% glucose and 0.2 M sodium phosphate buffer (pH6.0), and 3% (1S, 2S)-2-(N-ethylamino)-1-phenyl-1-propanol (EAM) wascontained in the reaction as substrate. A synthesis method for EAM isdescribed in J. Am. Chem. Soc., Vol. 50, pp. 2287-2292, 1928. Thereaction solution was incubated with shaking at 30° C. for 16 hours.Confirmation of (1S, 2S)-2-(N-ethylamino)-1-phenyl-1-propanol (EPE),which was β-aminoalcohol as the reaction product, was accomplished byHPLC. The column used was an Inertsil Ph-3 3.0×75 mm, the eluent wasaqueous 7% acetonitrile and 0.05 M sodium phosphate buffer (pH 6.0), andthe detection was carried out with UV (220 nm).

The results of the activity assay carried out in this manner are shownin Table 6. The pRET1104-introduced recombinant cells lacking theexogenous promoter region exhibited about the same activity as thepRET1102-introduced recombinant cells lacking the mak gene used as thecontrol, and no recombinant enzyme expression was found.

With transformation of pRET1114 into MAK-34 strain, high specificactivity was found compared to pRET1104. This indicated that thepRET1200 repA promoter region inserted into the vector functions as apromoter.

With transformation of pRET1138, the specific activity of therecombinant R. erythropolis MAK-34 strain was 37.7 μg/h·mL/O.D. whilethe specific activity of the recombinant R. erythropolis JCM2895 was34.9 μg/h·mL/O.D., and therefore expression of the enzyme in R.erythropolis strain was confirmed.

TABLE 6 Vector MAK-34 JCM2895 pRET1102 1.0 1.0 pRET1104 0.7 2.0 pRET111417.2 not tested pRET1138 37.7 34.9 

Specific activity (units: μg/h·mL/O.D.)

Example 13 Purification of Enzyme

The recombinant cells obtained in Example 12 were cultured at 30° C. for4 days in 100 mL of LB medium containing 100 μg/mL kanamycin, the cellswere harvested by centrifugation at 12,000 rpm for 5 minutes and theprotein having His-tag was purified with The QIAexpressionist Kit(Qiagen). Specifically, the cells were disrupted by ultrasonictreatment, the supernatant was obtained by centrifugation, and theprotein was purified with a nickel chelate column. Upon applying theobtained protein to SDS-PAGE, a band of protein, which molecular weightis approximately 28,000, was observed. This molecular weight is roughlyequivalent to the molecular weight of the aminoketone asymmetricreductase described in International Patent Publication WO02/070714,thus indicating that the aminoketone asymmetric reductase was producedin the recombinant Rhodococcus strains.

Example 14 Enzymatic Production of β-aminoalcohol

A 0.5 mL portion of reaction solution containing the purified enzyme(0.5 μg/mL) obtained in Example 13, 5 mM NADPH, 120 mM Tris-HCl (pH 7.5)and 5 mM EAM was reacted at 37° C. for 16 hours. The substrate andproduct (EPE) were analyzed by HPLC. The column used was an InertsilPh-3 3.0×75 mm, the eluent was aqueous 7% acetonitrile and 0.05 M sodiumphosphate buffer (pH 6.0), and the detection was carried out with UV(220 nm). The results confirmed production of EPE.

Similarly, the purified enzyme or the crude enzyme extract obtained fromthe recombinant cells cultured as described in Example 13 was reactedwith 1-2-dimethylaminopropiophenone and 1-amino-2-butanone, etc. andproduction of the corresponding β-aminoalcohols was confirmed.

INDUSTRIAL APPLICABILITY

As explained above, the plasmids and shuttle vectors of the inventionare derived from Rhodococcus strains (especially Rhodococcuserythropolis and Rhodococcus rhodnii), and when utilized them formodification of the same bacteria by recombination, they allow creationof bacterial strains that more efficiently produce aminoketoneasymmetric reductases. They also permit mass production of usefulenzymes including aminoketone asymmetric reductases in transformants.

1. An isolated DNA fragment having the nucleotide sequence set forth asSEQ ID NO:
 35. 2. An isolated plasmid or an isolated partial DNAfragment thereof, comprising a DNA replication region having thenucleotide sequence set forth as SEQ ID NO:
 35. 3. An isolated plasmidor an isolated partial DNA fragment thereof, comprising an ORF of SEQ IDNO: 1, 4, 5, 17, 22, 27, 29, 31 or 33 and a DNA replication regionhaving the nucleotide sequence set forth as SEQ ID NO:
 35. 4. Anisolated plasmid or an isolated partial DNA fragment thereof, comprisingan ORF of SEQ ID NO: 1, 4, 5, 17, 22, 27, 29, 31 or 33, a DNAreplication region having the nucleotide sequence set forth as SEQ IDNO: 35, and a promoter region.
 5. An isolated circular plasmidcomprising the DNA fragment according to claim 1 and one or morerestriction endonuclease cleavage sites selected from the groupconsisting of BamH I, EcoR I, Kpn I, Pvu II, Sac I, and Sma I, whereinthe size of the isolated circular plasmid is approximately 5.4 kbp. 6.An isolated shuffle vector replicable in bacteria belonging to the genusRhodococcus and E. coli, and comprising the DNA fragment according toclaim 1 and a DNA region replicable in E. coli.